A Novel Propane Monooxygenase Initiating Degradation of 1,4

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Letter

A Novel Propane Monooxygenase Initiating Degradation of 1,4-Dioxane by Mycobacterium dioxanotrophicus PH-06 Daiyong Deng, Fei Li, and Mengyan Li Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.7b00504 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 3, 2017

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Environmental Science & Technology Letters

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A Novel Propane Monooxygenase Initiating

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Degradation of 1,4-Dioxane by Mycobacterium

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dioxanotrophicus PH-06

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Daiyong Deng, Fei Li, Mengyan Li*

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Department of Chemistry and Environmental Science, New Jersey Institute of Technology,

7

Newark, NJ, USA 07102

8 9

*Address correspondence to Dr. Mengyan Li ([email protected])

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Phone: +1-973-642-7095

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Fax: +1-973-596-3586

1 ACS Paragon Plus Environment


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Abstract

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Monitored natural attenuation and bioremediation are cost-efficient and environment-

14

friendly approaches to mitigate prevalent 1,4-dioxane (dioxane) plumes. Unfortunately, their field

15

applications have been greatly undermined given our scarce knowledge of the diversity of dioxane

16

biodegradation pathways and associated key enzymes. At present, only tetrahydrofuran

17

monooxygenases (THF MOs) are known to initiate dioxane degradation in dioxane metabolizers.

18

In this study, we deciphered the essential catalytic role of a novel propane MO (encoded by the

19

prmABCD gene cluster) in dioxane metabolism by Mycobacterium dioxanotrophicus PH-06. This

20

propane MO is phylogenetically distinct from THF MOs based on the low amino acid sequence

21

identities (< 40 % for alpha subunits). Reverse transcription PCR analysis revealed that the

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prmABCD gene cluster is an intact transcription unit inducible by dioxane, THF, or propane.

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Further, biotransformation activity of this propane MO towards dioxane, THF, and propane was

24

confirmed using heterologous expression. Detection of 2-hydroxyethoxyacetic acid in the

25

expression clones proves that this propane MO catalyzes dioxane decomposition via α-

26

hydroxylation. This first enzymological identification of the propane MO in PH-06 expands our

27

understanding of dioxane metabolic pathways and unequivocally enables the development of

28

molecular tools to improve the assessment of natural attenuation and bioremediation at dioxane-

29

impacted sites.

30

Keywords:

31

Mycobacterium dioxanotrophicus PH-06, heterologous expression

1,4-dioxane,

propane

monooxygenase,

soluble


di-iron

monooxygenase,


prmABCD in Mycobacterium dioxanotrophicus PH-06

Mycobacterium smegmatis mc2-155

Propane MO

Biomass + CO2

Negative Control

pTip-QC2

pTip-prmABCD

Propane MO Expressing Treatment

32

1,4-Dioxane Concentration

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Negative Control

Propane MO Expressing Treatment

Incubation Time



33

1. Introduction

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1,4-Dioxane (dioxane) has emerged as a water contaminant of growing attention1, 2 given

35

its human carcinogenicity3 and prevalent occurrence4, 5. As a stabilizer for chlorinated solvents,

36

particularly 1,1,1-trichloroethane, dioxane has been detected as a frequent co-contaminant at

37

thousands of solvent-contaminated sites.2, 5, 6 Unfortunately, dioxane’s hydrophilic nature and low

38

KOC preclude the effective treatment by adsorption, air stripping, and other conventional

39

approaches.1, 2 Thus, the majority of the ongoing site remediation efforts primarily rely on pump

40

and treat in combination with chemical oxidation processes (e.g., hydrogen peroxide with ozone

41

or UV light).7, 8

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Monitored natural attenuation (MNA) and bioremediation are among the most economical

43

and eco-friendly treatment alternatives that are particularly suited for mitigating large and dilute

44

dioxane plumes.9 A recent data mining study provided evidence of dioxane attenuation at a

45

significant number of sites that were investigated based on their historical monitoring records.10

46

Further, occurrence and acclimation of naturally-occurring dioxane degraders at several dioxane-

47

impacted sites have been demonstrated using an array of molecular biological tools, including

48

quantitative PCR (qPCR), microarray, and DGGE.9, 11, 12 However, application and evaluation of

49

MNA and bioremediation have been substantially hurtled by our scare knowledge of the molecular

50

basis of dioxane biodegradation. Therefore, to develop feasible biomarkers and other molecular

51

tools with high specificity and profound monitoring value, it is of significant research priority to

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untangle enzymes in charge of the key steps of dioxane degradation.

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In recent years, over ten dioxane degrading bacteria have been isolated.13-21 However,

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tetrahydrofuran monooxygenases (THF MOs) are so far the only type of bacterial enzymes

55

identified in dioxane metabolizers that can initiate the biodegradation of this compound.22-25 Four


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THF MO-encoded gene clusters (thmADBC) have been reported in the archetypic dioxane

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degrader, Pseudonocardia dioxanivorans CB119013, 25-27, and three other Pseudonocardia and

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Rhodococcus species22-24. THF MOs are responsible for inserting a hydroxyl group at the α carbon

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position of dioxane leading to the subsequent cleavage of the high-energy ether bond (so-called

60

“α-hydroxylation”).25, 28, 29 Based on the phylogenetic analysis, THF MOs belong to a multi-

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component bacterial enzyme family named as soluble di-iron monooxygenases (SDIMOs).30-32

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SDIMOs are known for their versatile degradation capabilities and can be divided into six

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subgroups based on their substrate preference, sequence similarity, and gene component

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arrangement.31-33 THF MOs are categorized as the group-5 SDIMOs.

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Recent findings in pure strains16,

34

or enriched consortia35 revealed that dioxane

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metabolism may not always be limited to group-5 THF MOs. Notably, a propane MO encoded by

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the gene cluster prmABCD was postulated to be associated with dioxane degradation in

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Mycobacterium dioxanotrophicus PH-06, since (1) the monohydroxylated product of dioxane, 1,4-

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dioxane-2-ol, was detected as a metabolic intermediate14, and (2) all individual gene components

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were upregulated by the exposure of dioxane36. This propane MO in PH-06 belongs to the group-

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6 SDIMOs and exhibits low sequence identities (i.e., < 40%) with THF MOs (Table 1) and the

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putative group-5 propane MO (Table S1) in Rhodococcus jostii RHA137, an actinomycete that

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degrades dioxane after pre-grown with propane38, despite sharing the same four key enzyme

74

components. These results suggest that intrinsic dioxane attenuation potential is probably being

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underestimated using molecular assessments that are limited to THF MOs and their genes. Though

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previous intermediate analysis and expression assays shed light on the dioxane degradation process

77

in PH-06, no enzymatic evidence has been provided to unequivocally identify the key enzyme that

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initially attacks dioxane. This effort is urgently needed to prevent unspecific design of monitoring



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tools that potentially mistarget enzymes/genes involved in the conversion and assimilation of

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dioxane’s metabolites.

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In this study, we uncover the critical enzyme that initiates the oxidation of dioxane in PH-

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06 using heterologous expression, which has yet been done for any group-6 SDIMOs. Thus, we

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first evaluate the transcription pattern of the propane MO gene cluster in PH-06 to investigate the

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components needed for the proper expression of this enzyme. Oxidation activities and products of

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this propane MO are further assessed in the expression host to inevitably elucidate its catalytic

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function. This research advances our fundamental knowledge of dioxane metabolic processes and

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enables the development of molecular tools that amend current assessments of MNA and

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bioremediation potentials at contaminated fields.

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2. Materials and Methods

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2.1. Bacterial Strains

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Bacterial strains used in this study include Mycobacterium dioxanotrophicus PH-0614,

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Mycobacterium smegmatis mc2-155 (a highly electrotransformable mutant of ATCC-60739) as the

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heterologous expression host40, 41, and Escherichia coli DH5α (New England BioLabs, Ipswich,

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MA) as the cloning host. PH-06 was cultured in Ammonium Mineral Salts (AMS) medium with

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dioxane as the sole growth substrate42. mc2-155 and DH5α were grown in Luria–Bertani (LB)

96

medium.

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2.2. Gene Transcription and Expression Test

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To discern the transcription of the propane MO gene cluster induced by various substrate

99

compounds, triplicate treatments were prepared in 160-mL serum bottles containing 20 mL of

100

AMS medium and PH-06 inoculum. Dioxane, THF, 1-propanol, 2-hydroxyethoxyacetic acid

101

(HEAA), pyruvate, glucose, or succinate was amended as the sole carbon source to achieve an


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initial concentration of 2 mM. A parallel treatment was amended with propane in headspace (1.5 %,

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v/v). All treatments were incubated at 30 °C while shaking at 120 rpm. PH-06 cells at exponential

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phase (when half of the added substrates were consumed) were harvested for total RNA extraction

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and subsequent reverse transcription PCR (RT-PCR) and qPCR (RT-qPCR) analysis43 (see

106

Supporting Information).

107

2.3. Heterologous Expression of the PH-06 Propane MO

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A 4.0-kb fragment of the gene cluster prmABCD in PH-06 was amplified and cloned into

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the vector pTip-QC244 via restriction enzyme digestion and ligation, resulting in the appropriate

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recombinant construct designated as pTip-prmABCD. Plasmid pTip-prmABCD or empty vector

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pTip-QC2 was used to transform electrocompetent mc2-155 cells based on the method of Ly et

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al.40. After screening with chloramphenicol, successful transformant cells containing the plasmid

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pTip-QC2 constructs with and without the prmABDC insert [designated as mc2-155(pTip-

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prmABCD) and mc2-155(pTip-QC2), respectively] were cultured and dosed with thiostrepton to

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induce the heterologous expression prior to the following biotransformation assays. Production of

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the PH-06 propane MO components in induced mc2-155 transformants was examined by

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SDS/PAGE analysis. Details regarding the heterologous cloning and expression procedures are

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provided in the Supporting Information.

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2.4. Biotransformation Assays of the prmABCD Expressing Clones

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To perform oxidation assays, mc2-155(pTip-prmABCD) and mc2-155(pTip-QC2) cells

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were suspended in PBS buffer after induction25, 26, 45. The transformation of dioxane, dioxane-d8,

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THF, and propane were tested in triplicate, in 25-mL serum vials containing 4.5 mL of PBS buffer

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and 0.5 mL of cell suspensions. The initial biomass was estimated as 1.5 mg of total protein per

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vial using the Bradford assay46. Initial concentrations of dioxane, dioxane-d8, and THF were 0.61



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mM, 0.62 mM and 0.50 mM, respectively. Propane was added as neat amount of 28.6 µmol in the

126

headspace. Abiotic controls were prepared without cell suspensions. Treatments were all incubated

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at 30 ºC while shaking at 175 rpm. At the selected incubation time, aqueous (600 μL) or headspace

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(100 μL for propane) samples were removed and analyzed for the disappearance of the amended

129

compounds and production of metabolites (e.g., HEAA) by GC-FID or GC/MS analysis47, 48 (see

130

Supporting Information).

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3. Results and Discussion

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3.1. Dioxane-induced Polycistronic Transcription of the prmABCD Gene Cluster

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As evident by the visible bands of A, AB, BC, CD, and ABCD by RT-PCR analysis (Figure

134

1), all four prm genes encoding the propane MO were co-transcribed to produce a polycistronic

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mRNA transcript when PH-06 was exposed to dioxane, THF, and propane. However, no

136

transcription of these four prm genes was observed when PH-06 was fed with known metabolites

137

of dioxane, THF, and propane (e.g., HEAA25, 48, succinate22, and 1-propanol49) or other common

138

substrates (e.g., glucose and pyruvate). Similar results were obtained by RT-qPCR analysis (Figure

139

S2). After normalized with the treatment in which PH-06 was fed with pyruvate, the expression of

140

the prmA gene was significantly induced by dioxane, THF, and propane, but not by HEAA or 1-

141

propanol. The upregulation of the prmA gene was of comparable levels for dioxane (7.8 ± 1.3

142

folds), THF (7.4 ± 1.2 folds), and propane (7.4 ± 1.1 folds), which is in accordance with the RNA-

143

seq and RT-qPCR analysis by He et al.36.

144

Adjacent to this prmABCD cluster, there are no other genes annotated on the same strand

145

except a downstream groEL gene (Figure 1A and S3A). Previous molecular studies revealed the

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critical role of groEL-encoded chaperonin protein in the productive folding of SDIMO

147

hydroxylase subunits50, 51. However, transcription of this downstream groEL gene is independent


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from the prmABCD gene cluster, because (1) no DE or ABCDE transcript band was visible by the

149

RT-PCR analysis when the prmABCD gene cluster was induced by dioxane, THF, or propane

150

(Figure 1B), and (2) inverted sequence repeats exist at the immediate downstream of prmD (Figure

151

S5) indicating a putative rho-independent transcription termination site for the prmABCD gene

152

cluster. This confirms the prmABCD gene cluster as an intact polycistronic transcription unit,

153

allowing the subsequent heterologous expression assay.

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PH-06’s prmABCD gene cluster is carried by a composite transposon, which is bounded

155

by two insertion sequences (ISs) belonging to the family of IS256 (Figure S3). These two IS256

156

elements are replicas, though the one downstream of prmABCD (i.e., IS256-R) has been partially

157

chopped by latter gene disruption events (Figure S3 and S4). As IS256 transposases mediate gene

158

transposition via the conservative cut-and-paste mechanism52, 53, relocation of this prmABCD gene

159

cluster from chromosome to a plasmid promotes intercellular spreading of this unique catabolic

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advantage via horizontal gene transfer in environments where a selective pressure of dioxane

161

contamination is present.

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3.2. Oxidation of Dioxane by the PH-06 Propane MO in Heterologous Expression Clones

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To verify the role of this PH-06 propane MO in dioxane degradation, the prmABCD gene

164

cluster was cloned and expressed in mc2-155 as the heterologous host, because (i) it belongs to the

165

same Mycobacterium genus as PH-06, (ii) it is unable to oxidize or transform test substrates

166

(dioxane, THF, and propane54), and (iii) it was successfully used to express an analogous multi-

167

component group-3 SDIMO gene (smoXYB1C1Z) from Mycobacterium chubuense NBB441. As

168

depicted in Figure S6, SDS-PAGE analysis indicated successful heterologous expression of all

169

four propane MO components in the soluble protein fraction of the induced mc2-155(pTip-



170

prmABCD) cells in comparison with the control cells with the empty vectors [i.e., mc2-155(pTip-

171

QC2)].

172

Oxidation assays using mc2-155(pTip-prmABCD) cells demonstrated the produced

173

propane MO is capable of degrading dioxane, THF, and propane (Figure 2), which was also

174

verified with dioxane-d8 as a deuterated control. For the first two hours, instant oxidation rates of

175

dioxane, THF, dioxane-d8, and propane were estimated as 0.29, 0.54, 0.13, and 0.20 µmol

176

substrate/mg protein/hr, respectively. In contrast, no degradation activity of mc2-155(pTip-QC2)

177

transformants was observed towards any of the four tested substrates. No significant loss of

178

dioxane, THF, dioxane-d8, or propane was distinguished in the abiotic controls.

179

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Mycobacterium sp. ENV421 also harbors a group-6 SDIMO gene cluster45 that shares a

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close phylogenetic relationship with the prmABCD in PH-06 (amino acid sequence identity of 59.3%

181

for alpha subunits). Notably, ENV421 can cometabolize dioxane following the growth on propane.

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Unfortunately, the heterologous expression of this ENV421 group-6 SDIMO was unsuccessful

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possibly because only a portion of its gene cluster (~ 2.7 kb) was sequenced and cloned.45 It is

184

plausible to postulate that this ENV421 group-6 SDIMO is in charge of the oxidation of both

185

propane and dioxane, since the involvement of other MOs (e.g., a CYP153-type cytochrome P450

186

oxygenase and an AlkB-type alkane MO) in this strain was precluded based on enzymatic assays45.

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3.3. Detection of HEAA as a Dioxane Degradation Intermediate

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HEAA and 1,4-dioxane-2-one (PDX) are commonly detected as the terminal metabolites

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of dioxane in cells heterologously expressing SDIMOs25 or accumulated in bacterial

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cometabolism28, 48. To further investigate the metabolic products of the PH-06 propane MO in the

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heterologous expression cells, acidification was employed to distinguish the detection of HEAA

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and PDX. Figure 3 depicts that PDX was only observed when the filtered medium of dioxane-


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grown mc2-155(pTip-prmABCD) transformants was acidified by formic acid. However, without

194

acidification, no PDX was detected. A similar observation was verified when the induced

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transformants were exposed to dioxane-d8 (Figure 3). As HEAA converts to PDX after

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acidification55, our mass spectrometry results demonstrated that dioxane was transformed to

197

HEAA, but not PDX, in heterologous clones expressing the PH-06 propane MO. The detection of

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HEAA corroborates our hypothesis that this propane MO activates the α-carbon of dioxane to

199

insert a hydroxyl group and form 1,4-dioxane-2-ol14, which is subsequently oxidized to HEAA.

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This is similar to the initial biotransformation pathways observed in wildtype or transformant cells

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expressing group-5 THF MOs.22, 25, 28, 29, 48

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Overall, we have identified a novel group-6 propane MO in PH-06 and proved its catalytic

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function of initiating the oxidation of dioxane via α-hydroxylation. As previous research regarding

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dioxane biodegradation has been extensively centered on group-5 THF MOs, this is the first study

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that has uncovered a bacterial dioxane-degrading MO beyond this specific enzyme group through

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enzymological analysis. This study also provides the first demonstration of successful

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heterologous expression of group-6 SDIMOs, allowing future kinetic and structural investigations

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at the enzymatic level. Our findings extend our understanding of the diversity of dioxane degrading

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oxygenases and avail us with a novel enzyme for mitigating dioxane contamination, as well as

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function-characterized sequences for developing molecular tools to improve the evaluation of

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natural attenuation and bioremediation at dioxane-impacted sites.

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Acknowledgements

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This project was supported by the start-up fund from the Department of Chemistry and

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Environmental Science at New Jersey Institute of Technology (NJIT), USGS WRRI Program

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(#2017NJ388B), NJIT Faculty Seed Grant (#211247) and Undergraduate Research and Innovation



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(URI) Program. We thank Dr. Yoon-Seok Chang (POSTECH), Dr. Nicolas Coleman (Sydney

217

University), Dr. Tomohiro Tamura (AIST) for providing experimental strains and vectors. We also

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thank Dr. John Wilson (Scissortail Environmental Solutions, LLC) for his insightful comments.

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The authors declare no competing financial interest.


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(A)

prmA

prmC

A

B

C

D

prmB A

E

CD

Succinate Glucose

702 bp

Pyruvate

1072 bp

HEAA

BC

1-Propanol

948 bp

DE ABCD

Propane 1936 bp

THF Dioxane

2095 bp

ABCDE

220 221 222 223 224 225 226 227

(B) Genome DNA

prmD

254 bp AB

groEL

A

3640 bp

AB

BC

CD

DE

ABCD ABCDE 16S

Figure 1. Transcription of the prmABCD gene cluster when PH-06 was fed with dioxane, THF, propane, and other substrates as sole carbon and energy sources. Positions and sizes of the targeted transcript fragments are depicted in scale (A). RT-PCR products representing induced production of the targeted mRNA transcripts are visualized as bands of their accordant fragment sizes via gel electrophoresis (B). The 16S rRNA gene of PH-06 was employed as a positive expression control gene for all treatments. Genomic DNA was used as the positive template among PCR reactions. Original gel electrophoresis images are provided in Figure S1.



0.8

pTip-QC2

(A)

0.6

0.4

0.2

Abiotic Control

0.8

THF Concentration (mM)

Dioxane Concentration (mM)

pTip-prmABCD

0.0

(B)

0.6

0.4

0.2

0.0 0

5

10

15

20

0

5

(C)

0.8

0.6

0.4

0.2

0.0 0

229 230 231 232

5

10

10

15

20

Time (h)

15

Propane Concentration (mM)

Dioxane-d8 Concentration (mM)

Time (h)

228

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20

(D)

1.0

0.9

0.8

0.7

0.6 0

Time (h)

5

10

15

20

Time (h)

Figure 2. Degradation activity of heterologous PH-06 propane MO expression clones towards (A) dioxane, (B) THF, (C) dioxane-d8, and (D) propane. Substrate removal was compared among mc2155 transformant clones containing plasmid pTip-prmABCD, the empty vector of pTip-QC2, and abiotic control. Error bars indicate standard deviations among triplicates.


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(A)

HEAA with Acidification Metabolites of Dioxane without Acidification Metabolites of Dioxane with Acidification Metabolites of Dioxane-d8 without Acidification Metabolites of Dioxane-d8 with Acidification

(B)

PDX-d6 PDX (C)

233 234 235 236 237 238

Figure 3. Detection of metabolic products of dioxane in mc2-155 clones expressing the PH-06 propane MO. (A) Chromatographs of GC/MS analysis of dioxane and dioxane-d8 metabolites with and without the acidification of formic acid. Acidified HEAA was employed as a positive control. Positive chromatographic peaks of PDX and PDX-d6 are highlighted in (A) and their mass spectra are presented in (B) and (C), respectively.



239 240 241

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Table 1. Comparison between the essential enzymatic protein components of the group-6 propane MO in Mycobacterium dioxanotrophicus PH-06 and the group-5 THF MO in Pseudonocardia dioxanivorans CB1190. Group-6 Propane MO in PH-06 Key Enzyme Components

Group-5 THF MO in CB1190

Amino Acid Identity

Gene Name

Amino Acid Residues

Molecular Mass (kDa)

Gene Name

Amino Acid Residues

Molecular Mass (kDa)

prmA

513

58.9

thmA

545

62.5

39.6%

prmB

364

40.2

thmB

346

39.3

29.1%

Coupling protein

prmC

106

11.9

thmC

117

12.6

28.1%

Reductase

prmD

344

37.3

thmD

361

40.0

34.3%

Hydroxylase alpha subunit Hydroxylase beta subunit

242


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Supporting Information Available:

244 245 246 247 248

Detailed experimental procedures for RNA extraction, RT-PCR, RT-qPCR, cloning and heterologous expression, SDS-PAGE, and analytical methods; comparison between propane MOs in PH-06 and RHA1; genetic characterization of the PH-06 prmABCD gene cluster and the associated composite transposon; additional results of transcription analysis and induced protein production verified in expression clones.



249

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A Novel Propane Monooxygenase Initiating Degradation of 1,4

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