Volatile Gas Production by Methyl Halide Transferase: An In Situ

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Volatile gas production by methyl halide transferase: an in situ reporter of microbial gene expression in soil Hsiao-Ying Cheng, Caroline A. Masiello, George N. Bennett, and Jonathan J. Silberg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01415 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

Environmental Science & Technology

1 2 3 4 5 6 7 8

Volatile gas production by methyl halide transferase: an in situ

9

reporter of microbial gene expression in soil

10 11

Hsiao-Ying Cheng1, Caroline A. Masiello2,3, George N. Bennett3,4, and Jonathan J.

12

Silberg1, 3, *

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Author affiliations: 1. Department of Bioengineering, Rice University, 6100 Main Street, MS 142, Houston, TX 77005 2. Department of Earth Science, Rice University, 6100 Main Street, MS 126, Houston, TX 77005 3. Department of Biosciences, Rice University, 6100 Main Street, MS 140, Houston, TX 77005 4. Department of Chemical and Biomolecular Engineering, 6100 Main Street, MS 362, Houston, TX 77005

*Address correspondence to: Dr. Jonathan Silberg 6100 Main Street, Houston, TX 77005 Phone: 713-348-3849; Fax: 713-348-5154 Email: [email protected]

33 34 35

Keywords: conjugation, dynamics, gene expression, horizontal gene transfer, methyl

36

halide transferase, reporter, soil, synthetic biology, water holding capacity.

1

ACS Paragon Plus Environment


37

ABSTRACT

38

Traditional visual reporters of gene expression have only very limited use in soils

39

because their outputs are challenging to detect through the soil matrix. This severely

40

restricts our ability to study time-dependent microbial gene expression in one of the

41

Earth’s largest, most complex habitats. Here we describe an approach to report on

42

dynamic gene expression within a microbial population in a soil under natural water

43

levels (at and below water holding capacity) via production of methyl halides using a

44

methyl halide transferase. As a proof-of-concept application, we couple the expression

45

of this gas reporter to the conjugative transfer of a bacterial plasmid in a soil matrix and

46

show that gas released from the matrix displays a strong correlation with the number of

47

transconjugant bacteria that formed. Gas reporting of gene expression will make

48

possible dynamic studies of natural and engineered microbes within many hard-to-

49

image environmental matrices (soils, sediments, sludge, and biomass) at sample scales

50

exceeding those used for traditional visual reporting.

51

2


Page 2 of 42

Page 3 of 42


52

INTRODUCTION

53

Cells are routinely programed to report on their behaviors by producing molecular

54

outputs that can be visually quantified, such as colored1, fluorescent2 and luminescent

55

molecules3. These reporters transformed biology by enabling the real-time tracking of

56

dynamic biological processes, such as cell localization and migration4, enzyme

57

activation5, macromolecular interactions6, and horizontal gene transfer7.

58

Simultaneously, innovations in DNA sequencing8, microchip biotechnologies9, and mass

59

spectrometry10 transformed our understanding of complex microbial communities by

60

enabling us to catalog what organisms are present in a soil11, including microbes we

61

cannot yet cultivate, and the ensemble of RNA, proteins, and metabolites synthesized

62

by communities. This explosion of microbiome data has generated many hypotheses

63

about the connections between soil microbial behaviors and ecosystem-scale

64

phenomena. These hypotheses would be easy to test in a transparent matrix using the

65

palette of colored reporter proteins. However, soils and sediments display high

66

absorbance at the wavelengths of light required for visual reporting, restricting the

67

application of existing gene expression reporters to organisms in specialized

68

microcosms that are compatible with imaging or cells extracted from soils12,13.

69

We sought to develop a reporting approach that could be used nondisruptively to

70

study microbial gene expression in bulk soils under conditions that reflect those

71

commonly used for laboratory soil incubations14-16. We hypothesized that methyl halide

72

transferases (MHT) could provide real-time, nondestructive reporting of microbial

73

processes within hard-to-image matrixes like soils and sediments (Fig. 1a). MHT

74

synthesize volatile methyl halides (CH3X) using halide ions (Cl-, Br-, or I-) and S-

75

adenosyl methionine, a common cellular metabolite17. The initial discovery and 3



76

characterization of an MHT suggested that these enzymes might be well suited as

77

microbial reporters in hard-to-image conditions like soils because they synthesize a

78

volatile gas that is easy to detect using gas chromatography-mass spectrometry (GC-

79

MS)18. Early work showed that MHT are encoded by a single polypeptide and

80

demonstrated that these enzymes can be produced in a functional form in non-native

81

bacteria like Escherichia coli18,19. Since the detection of MHT in algae, fungi, and

82

plants20, a range of organisms and soils have been found to encode MHT and produce

83

this volatile metabolite21-25. While CH3X are produced by soil microbes and plants18-25,

84

these gases are expected to be present at very low levels in autoclaved soils. For this

85

reason, we hypothesized that microbes introduced into autoclaved soils could be

86

programmed using synthetic biology26-28 to report on promoters whose activities change

87

dynamically with time and soil conditions.

88

Here we describe how an MHT-expressing E. coli can be used to report on gene

89

expression in a microbe within a moist soil under a range of growth conditions in a lab

90

setting. In proof-of-concept experiments, we used this gas reporting approach to

91

monitor E. coli conjugation within an agricultural soil and examined how hydration

92

affects horizontal gene transfer. These experiments demonstrate that gas reporting can

93

be used to non-disruptively monitor a dynamic microbial behavior within natural soil

94

conditions.

95

4


Page 4 of 42

Page 5 of 42


96

METHODS

97

Materials. Enzymes for DNA manipulation were from New England Biolabs and

98

ThermoFisher, synthetic oligonucleotides and genes were from Integrated DNA

99

Technologies and nucleotides for PCR were from Roche. Bacterial growth medium

100

components were molecular biology grade and from Sigma-Aldrich and Amresco.

101

Antibiotics were from Research Products International. Kits for DNA and plasmid

102

purification were from Zymo Research and Qiagen, respectively.

103

Bacterial Growth Medium. Lysogeny Broth (LB) and LB-agar plates containing the

104

indicated levels of antibiotics were used to amplify strains and plasmids. M63 medium, a

105

defined growth medium with low osmolarity that lacks halide ions, was used in cultures

106

studying cellular gas production. To create M63 medium for studying the effects of

107

halide salt concentration on gas production, we diluted a M63 salt stock (5x), 1 M

108

MgSO4 (1000x), 20% glucose (100x), 0.5% thiamine (10,000x), 20% casamino acids

109

(200x), and 4M sodium halide ions (varying dilutions) into autoclaved water. The M63

110

salt stock (75 mM ammonium sulfate, 0.5 M potassium phosphate monobasic, and 10

111

µM ferrous sulfate) was adjusted to pH 729. All soil measurements were performed by

112

hydrating soil to the indicated levels using 1x M63 medium containing 100 mM NaBr.

113

Plasmids. All PCR amplicons used for vector assembly were synthesized using

114

Phusion® High-Fidelity DNA polymerase and a BIO-RAD T100 Thermocycler, while

115

Vent® DNA Polymerase was used for screening. A previously described KmR sender

116

plasmid30 was PCR amplified to create a linear vector that contains: (i) the Aequeora

117

victoria green fluorescent protein (GFP) gene, encoded by part number BBa_E0040

118

from the International Genetically Engineered Machine (iGEM) Foundation’s registry of

5



119

standard biological parts, under control of the lasR promoter (PlasR), and (ii) the

120

Pseudomonas aeruginosa LasR gene under control of a constitutive promoter. The

121

resulting amplicon was used in a Golden Gate assembly31 reaction with a commercially

122

synthesized Batis maritima MHT gene to create a plasmid (pHC.plas:MHT:GFP) having

123

a bicistronic message encoding MHT and GFP (see Supporting Information for

124

sequence). For this and all other Golden Gate assembly reactions, we mixed the DNA

125

being assembled with BsaI and T4 DNA ligase, and subjected the reaction to 25 cycles

126

of digestion (37°C) and ligation (16°C) to ensure high assembly efficiency. The product

127

of the Golden Gate reaction was transformed into E. coli XL1-Blue (Stratagene),

128

colonies were plated on LB-agar containing kanamycin, and plates were incubated

129

overnight at 37°C. Individual colonies were used to inoculate LB cultures, cultures were

130

growth overnight, and pHC.plas:MHT:GFP was obtained by purifying plasmid using a

131

QIAGEN MiniPrep Kit. In pHC.plas:MHT:GFP, MHT and GFP translation initiation are

132

regulated by strong RBSs, BBa_B0034 and BBa_B0030 (from the iGEM registry), and

133

their transcription is regulated by LasR, a transcriptional regulator that induces

134

expression from the PlasR when it binds to N-(3-oxodecanoyl)-L-homoserine lactone, an

135

acylhomoserine lactone (AHL)32.

136

A conjugative plasmid (pF-mht) was created by modifying the F-plasmid from E.

137

coli XL1-Blue using the Datsenko-Wanner method33 so that it lacks the tetracycline-

138

efflux pump (TetA) and contains a kanamycin-resistance cassette (KmR) and a MHT-

139

mCherry fusion protein. In pF-mht, MHT-mCherry was placed under control of a TetR-

140

repressible promoter (Ptet), so that expression is repressed by TetR but constitutive in

141

the absence of TetR. To create this plasmid, we PCR amplified separate DNA encoding

6


Page 6 of 42

Page 7 of 42


142

KmR, Ptet, BCD2 RBS34, and a MHT-mCherry gene fusion. Golden Gate was then used

143

to assemble these amplicons into a single linear DNA encoding a fusion of these

144

components. This DNA was designed to contain an additional 40 base pairs of

145

sequence at each end that is homologous to the regions of the F-plasmid that encode

146

the C-terminus of TetR and C-terminus of TetA. The linear DNA was electroporated into

147

E. coli XL1-Blue that contained pKD46, a plasmid that encodes the recombinase

148

RepA10133, and expression of RepA101 was induced with 0.2% arabinose to facilitate

149

recombination. Cells were grown on LB-agar plates containing 25 µg/mL of kanamycin

150

to obtain single colonies, and these colonies were then screened for: (i) sensitivity to 5

151

µg/mL tetracycline which arises from disruption of TetA, (ii) production of red

152

fluorescence arising from mCherry expression, and (iii) constitutive production of CH3Br.

153

E. coli XL1 Blue does not express TetR, so cells harboring the desired pF-mht

154

constitutively produce mCherry, MHT, and CH3Br. All plasmids were verified by

155

commercial DNA sequencing (Lone Star Labs)35.

156

Bacterial strains. E. coli XL1-Blue was used for molecular biology to construct and

157

amplify plasmids, and E. coli MG1655 was used as a control for all measurements

158

analyzing methyl halide production by cells lacking an MHT. An MHT-expressing E. coli

159

strain (MG1655-mht) was built using Clonetegration36, a one-step cloning and

160

chromosomal integration method. This was achieved by using Golden Gate to assemble

161

two amplicons generated by PCR, one encoding the B. maritima MHT gene and the

162

other encoding a previously described promoter-RBS fusion (P14:BCD2)34, cloning the

163

assembled DNA into the plasmid pOSIP-KO36 using Gibson Assembly37, transforming

164

the resulting plasmid (named pOSIP-KO-mht) into E. coli MG1655 using heat-shock,

7



165

and selecting for transformants on LB-agar plates containing 25 µg/mL of kanamycin. A

166

colony obtained from this selection, which had the DNA integrated into phage 186 attB

167

site within the chromosome, was transformed with the plasmid pE-FLP to remove the

168

KmR cassette. Chromosomal integration and KmR removal was verified by colony PCR

169

as previously described36.

170

An E. coli MHT-donor strain (MG1655-tetR) was created by chromosomally

171

integrating a spectinomycin resistant cassette (SpecR), the gene encoding tetracycline

172

repressor (TetR) and the gene encoding lac repressor (lacI) into the λ attachment site of

173

E. coli MG1655 using the λ recombination system38. An integration plasmid, pZS4Int-

174

lacI/tetR, from the pZ Expression System manufactured by Expressys, which

175

constitutively expresses TetR, was PCR amplified to introduce a second AvrII restriction

176

site adjacent to the pSC101 origin. The resulting plasmid was digested with AvrII to

177

remove the pSC101 origin, the remaining DNA fragment was circularized using T4 DNA

178

ligase, and this circular DNA (pZS4Int-lacI/tetR-AvrII) was transformed into MG1655

179

harboring the integrase-expressing plasmid pLDR838 using heat-shock. The resulting

180

cells were plated on LB-agar plates containing 50 µg/mL spectinomycin to select for

181

cells that contain the TetR gene integrated into the λ attachment site. Integration was

182

verified by PCR amplifying the TetR gene and visualizing the presence of TetR

183

amplicon using an agarose gel.

184

An E. coli recipient strain (MG1655-chl) was generated by chromosomally

185

integrating a chloramphenicol-resistant cassette (CmR) cassette into the phage 186 attB

186

site in E. coli MG1655 using Clonetegration36. This was achieved by PCR amplifying a

187

chloramphenicol-resistance cassette (CmR), digesting the product of this reaction and

8


Page 8 of 42

Page 9 of 42


188

pOSIP-KO using BamHI and SpeI, ligating the resulting DNA using T4 DNA Ligase,

189

transforming the resulting plasmid (named pOSIP-KO-Cm) into MG1655 using heat

190

shock, and plating the cells on LB-agar containing 25 µg/mL kanamycin and 25 µg/mL

191

chloramphenicol. Colonies obtained from this selection were transformed with pE-FLP

192

to remove KmR, and integration of the CmR at the 186 attB site was verified using colony

193

PCR36.

194

An E. coli transconjugant strain (MG1655-Tc) was generated by mating MG1655-

195

tetR and MG1655-chl. MG1655-tetR (105 colony forming units, cfu) transformed with pF-

196

mht was incubated with MG1655-chl (107 cfu) in LB medium (20 µL) for 4 hours at 37°C

197

without shaking, and plated on LB-agar containing kanamycin (25 µg/mL) and

198

chloramphenicol (17 µg/mL). After overnight growth at 37°C, colonies were visually

199

inspected to assess mCherry expression, and twenty colonies appearing red were used

200

to inoculate 1 mL M63 medium containing 100 mM NaBr, 25 µg/mL kanamycin and 17

201

µg/mL chloramphenicol. After 24 hours of growth in 2 mL gastight vials, CH3Br

202

production was analyzed using GC-MS (see Supporting Information Fig. S1) as

203

described below. A CmR and KmR transconjugant strain obtained from conjugation in

204

liquid culture, which displayed representative gas production, was used for all studies

205

comparing donor and transconjugant gas production.

206

GC-MS analysis of CH3X production. The analytical system consisted of an Agilent

207

7820 gas chromatography (GC) with DB-VRX capillary column (30m, 0.15 mm ID, and 1

208

µm film) and a 5977E mass spectrometry (MS). Samples were injected into the GC

209

using an Agilent 7693A liquid autosampler with either a 100 µL (Agilent G4513-80222)

210

or 250 µL gastight syringe (Agilent G4513-60560). The oven temperature was

9



211

programmed to hold at 45°C for 1 minute, followed by an increase to 60°C. The total run

212

time was 1 minute and 32 seconds. The 5977E MSD was configured for selected ion

213

monitoring mode, specifically to CH3Cl (MW = 50 and 52 for CH335Cl and CH337Cl,

214

respectively), CH3Br (MW = 93.9 and 95.9 for CH379Br and CH381Br, respectively), or

215

CH3l (MW = 127 and 142 for 127I and CH3127I). The peak area of a sample was

216

integrated using Agilent’s MassHunter Workstation quantitative analysis; the major ions

217

(CH335Cl, CH379Br, and CH3127I) were used for quantifying peak areas while the minor

218

ion was used as a qualifier. Standard curves for CH3Cl and CH3Br were obtain by

219

mixing methanol (100 µL) containing serial dilutions of analytical standards provided in

220

methanol (Sigma-Aldrich) with M63 medium (900 µL) containing either 100 mM NaCl or

221

NaBr. These mixtures were incubated in 2 mL gastight vials for 3 hours at 37°C with

222

shaking at 290 rpm to allow gas to equilibrate between the aqueous and gas phases,

223

and CH3X levels were measured within a 50 µL sample of the headspace gas. All

224

experiments except the conjugation measurements used a 100 µL syringe to inject and

225

analyze CH3X levels in 50 µL of headspace gas; the conjugation experiments used a

226

250 µL syringe to inject and analyze CH3Br levels in 125 µL of headspace gas. Our

227

standard curve for CH3Br revealed that our maximum signal from our longest

228

incubations (~10 ppm) was 103 lower than the level used for fumigation in agriculture

229

(~104 ppm)39,40 and higher than atmospheric background (~10-5 ppm)41. Our maximum

230

CH3Br signal observed in conjugation experiments was also lower than the levels of

231

methyl halides (CH3Cl and CH3I) previously found to strongly induce the expression of

232

the adaptive response protein (Ada) in E. coli, a microbial response that is caused by

233

the deleterious effects of methylation induced by these compounds42.

10


Page 10 of 42

Page 11 of 42

234


For soil experiments using varying amounts of water and soil, we calculated the total

235

mass of CH3Br in each vial using the standard curve and Henry’s law for adjusting the

236

gas partitioning between liquid and gas phase as previously described43. For this

237

calculation, we estimated the total CH3Br mass (W total) as the product of the

238

concentration measured in the gas phase (Cgas), the sum of the partition coefficient and

239

the ratio of the volume (K + Vgas/Vliquid), and the volume of the liquid phase, i.e., W total =

240

Cgas·(K + Vgas/Vliquid) ·Vliquid. The volume of the gas phase was estimated by subtracting

241

the volume of the liquid phase and the volume of the soil calculated by dividing soil

242

mass to its density (KBS top soil gravel free bulk density is reported as 1.5 g/ml) from

243

the total volume of the vial, i.e., Vgas = Vtotal – Vliquid – (W soil/ Dsoil).

244

Cell viability. A stationary phase culture of MG1655-mht (1 mL) was washed twice with

245

10% glycerol (ACROS, analytical chemistry grade) and resuspended in 10% glycerol to

246

an optical density (OD) of 0.05, measured at 600 nm. M63 medium (200 µL) containing

247

varying concentrations of halides (0.01 to 1600 mM NaCl, NaBr, or NaI) was arrayed in

248

a flat bottom 96-well plate, and each well was inoculated with 2 µL MG1655-mht from

249

the resuspended culture, and growth was assessed in unsealed wells. Growth was

250

continuously monitored at 37°C for 24 hours while shaking at 216 rpm with 1.5 mm

251

amplitude using a TECAN M1000 Pro Microplate Reader by measuring absorbance at

252

600 nm every five minutes. These OD values were fit to Equation 1:

253

OD = A / {1 + exp[(4µm/A)(λ – t) + 2]}

(Eq 1),

254

where t is time, λ represents the growth delay, µm is the maximum growth rate, and A

255

represents the asymptote and the maximum OD44. Data shown for each growth

256

experiment represents the average of three independent measurements. To examine

11



257

the effect of CH3Br accumulation on cell viability, MG1655-mht and its parental strain

258

were also incubated in sealed cuvettes using growth medium containing 10 g/L

259

tryptone, 5 g/L yeast extract, and 100 mM sodium bromide. Overnight cultures of E. coli

260

MG1655 and MG1655-mht presented no significant differences in cell growth under

261

these conditions.

262

Liquid culture analysis of CH3X production. Stationary phase MG1655-mht (10 µL)

263

that had been washed and resuspended in 10% glycerol was used to inoculate M63

264

medium (1 mL) containing varying concentrations of halides (0.01 to 1600 mM NaCl,

265

NaBr, or NaI). These halides are required as substrates for the MHT in addition to S-

266

adenosyl methionine, a metabolite that is synthesized from methionine and ATP45.

267

While methionine can be synthesized by E. coli, it was also provided in the growth

268

medium in the form of casamino acids. Each culture was incubated at 37°C in 2 mL

269

glass vials that were capped with gastight seals (Phenomenex) while shaking at 290

270

rpm. After 24 hours, CH3X levels were analyzed using GC-MS. In the case of

271

measurements comparing gas production from different titers of MG1655-mht, we first

272

estimated the cell number of the cultures using OD600 measurements, washed cells

273

twice with 10% glycerol and diluted cells to the indicated titer using 1 mL M63 medium

274

containing 100 mM NaBr. Each sample was placed in a 2 mL vial, crimped, and

275

incubated at 37°C. After 1 hour incubations, the headspace gas (125 µL) in each

276

sample was measured with the GC-MS. Data shown for each liquid culture experiment

277

represents the average of three independent measurements.

278

Anaerobic CH3X analysis. MG1655 transformed with pHC.plas:MHT:GFP was grown

279

to stationary phase in LB medium containing 50 µg/L kanamycin. This culture was

12


Page 12 of 42

Page 13 of 42


280

washed twice with 10% glycerol, and diluted to an OD600 = 0.05 in M63 medium

281

containing 100 mM NaBr and 50 µg/mL kanamycin. Cultures (5 mL) were grown in

282

capped Hungate vials (15 mL) containing or lacking 100 nM N-(3-oxodecanoyl)-L-

283

homoserine lactone, AHL (Sigma-Aldrich). Anaerobic cultures were generated by

284

purging the air with nitrogen gas in the Hungate vials using a vacuum gas manifold.

285

After incubating for 24 hours at 37°C while shaking at 250 rpm, CH3Br levels were

286

measured by manually injecting headspace gas into the GC-MS. GFP levels were

287

measured by uncapping vials, arraying 200 µL of each culture in a flat bottom 96-well

288

plate, and analyzing green fluorescence (λex = 488 nm; λem = 509 nm) and optical

289

density using a TECAN M1000 microplate reader. The small GFP induction in cells

290

derived from anaerobic cultures is attributed to a brief cellular exposure to air ( 0.05). Error bars represent ±1σ

790

calculated using 3 replicates.

791

Figure 3. Using CH3Br production to report on horizontal gene transfer. (A)

792

Scheme illustrating the genetic program used to couple MHT transcription and gas

793

production

794

chloramphenicol (gray) and plates containing both chloramphenicol and kanamycin

795

(blue) after growing mixtures of donor (~105 cfu) and acceptor (~107 cfu) cells in static

796

(B) liquid culture, (C) soil hydrated to 100% WHC, and (D) soil hydrated to 50% WHC.

797

The number of transconjugants obtained in liquid was significantly greater than that

798

obtained in soil at all time points except at 3 hours (two tailed t-test; p < 0.05). (E) The

799

conjugation frequency at each time was calculated as the ratio of transconjugants to

800

receiver cells measured using cfu from liquid cultures (white), soil hydrated to 100%

801

WHC (gray), and soil hydrated to 50% WHC (black). The conjugation frequency in soil

802

was significantly lower than in liquid culture at all time points exceeding 5 hours, and the

803

conjugation frequency in soil at 50% WHC was significantly lower than soil at 100%

804

WHC at the 2, 3, 8, 10, and 24 hour time points (two tailed t-test; p < 0.05). (F)

805

Headspace CH3Br measurements prior to cfu analysis is shown for each condition. The

806

gas production in soil was significantly lower than that in liquid culture at six time points

to

conjugation.

Soil-extracted

37

cfu

on


LB-agar

plates

containing


Page 38 of 42

807

(2, 3, 4, 6, 8 and 10 hours), and the gas production from soil at 50% WHC was

808

significantly lower than 100% WHC after 3 hours (two tailed t-test; p < 0.05). (G) Gas

809

production from soil is proportional to the number of extractable transconjugant cfu. A fit

810

of the data to the line log(Tc cfu) = 0.65 + 0.88·log(µg CH3Br/hr) yields a R value = 0.94.

811

Error bars represent ±1σ calculated using 3 replicates.

38


Page 39 of 42


264x264mm (72 x 72 DPI)



264x264mm (72 x 72 DPI)


Page 40 of 42

Page 41 of 42


264x264mm (72 x 72 DPI)



264x198mm (72 x 72 DPI)


Page 42 of 42

Volatile Gas Production by Methyl Halide Transferase: An In Situ

Recommend Documents