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FEMS Microbiology Letters 237 (2004) 355–361

A 19F NMR study of fluorobenzoate biodegradation bySphingomonas sp. HB-1

F.G. Hidde Boersma a, W. Colin McRoberts b, Steven L. Cobb c, Cormac D. Murphy a,*

a Department of Industrial Microbiology, University College Dublin, Belfield, Dublin 4, Irelandb Department of Agriculture and Rural Development for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK

c School of Chemistry, University of St. Andrews, Fife KY16 9ST, UK

Received 27 April 2004; received in revised form 27 June 2004; accepted 30 June 2004

First published online 14 July 2004

Abstract

While several microorganisms readily degrade 2- and 4-fluorobenzoates, only a very small number appear to catabolise the 3-

fluoro isomer, owing to the accumulation of toxic intermediates. Here we describe the isolation of a bacterium capable of using

3-fluorobenzoate as a sole source of carbon and energy, and the experiments conducted to define the steps involved in the biodeg-

radation of this compound. The organism was identified as a strain belonging to the genus Sphingomonas by sequence analysis of its

16S rRNA gene. To date no other organism from this genus is known to degrade this compound. Using fluorine nuclear magnetic

resonance spectroscopy (19F NMR) to analyse the culture supernatant it was possible to observe the disappearance of 3-fluor-

obenzoate and the appearance of fluoride ion and four other fluorinated compounds. These were identified as 3-fluorocatechol,

2-fluoromuconic acid and 3- and 5-fluoro-1,2-dihydro-1,2-dihydroxybenzoates. Thus, the likely catabolic pathway involves dioxy-

genation of 3-fluorobenzoate yielding fluorocatechol and subsequent intra-diol cleavage to yield fluoromuconic acid. The organism

can also use 2- and 4-fluorobenzoates as growth substrates.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Dioxygenation; Fluorobenzoate; 19F NMR

1. Introduction

The widespread use of organofluorine compounds by

the agrochemical, pharmaceutical and plastic industries

has resulted in these compounds becoming significant

environmental contaminants [1]. However, while much

is known about the microbial degradation of chlori-

nated organic compounds, our understanding of the

biodegradation of organofluorine compounds is poor.Progress in this area has improved with the emergence

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.06.052

* Corresponding author. Tel.: +353-1-716-1311; fax: +353-1-716-

1183.

E-mail address: cormac.d.murphy@ucd.ie (C.D. Murphy).

of 19F NMR as an analytical tool for studying the me-tabolism of fluorinated compounds [2,3].

The catabolism of fluorobenzoates has been studied

in severalmicroorganisms andbacteria have been isolated

that are capable of growing on 2- and 4-fluorobenzoate as

a sole carbon and energy source. 4-Fluorobenzoate typi-

cally undergoes dioxygenation to yield 4-fluorocatechol

followed by intra-diol cleavage forming 3-fluoro-cis,cis-

muconic acid. Cycloisomerization yields 4-fluoromuc-onolactone (4-carboxymethyl-4-fluorobut-2-en-4-olide)

and fluoride ion is eliminated forming maleylacetic acid,

which is channelled into the TCA cycle via oxoadipate

[4,5]. An alternative pathway has been found in anAureo-

bacterium sp., whereby the fluoride is enzymatically

removed in the initial step of degradation to yield

. Published by Elsevier B.V. All rights reserved.

356 F.G. Hidde Boersma et al. / FEMS Microbiology Letters 237 (2004) 355–361

4-hydroxybenzoate [6]. Dioxygenation of 2-fluorobenzo-

ate can result in either the spontaneous elimination of

fluoride forming catechol, or the formation of 3-fluoro-

catechol, which is a very poor substrate for catechol 1,2-

dioxygenases and can accumulate in, and poison, cells.

Nevertheless, there are some examples of bacteria usingthis compound as a growth substrate [7,8].

The co-metabolism of 3-fluorobenzoate has been re-

ported [9,10], but this compound appears to be a less ame-

nable substrate for microbial growth than the 2- or

4-fluoro isomers. A partially characterised strain (Agro-

bacterium-Rhizobium branch) isolated from a Nepalese

soil [11] was subjected to metabolic investigations and it

was concluded that the ability of amicroorganism to growon 3-fluorobenzoate was dependent upon the regioselec-

tivity of the initial dioxygenation reaction; however, only

fluoride ion and 2-fluoromuconic acid were identified in

the culture fluid in this study. Here we report the isolation

and characterisation of a bacterium capable of growing

on all three isomers of fluorobenzoate and the identifica-

tion of several catabolic intermediates by 19F NMR, al-

lowing elucidation of the degradative pathways.

2. Materials and methods

2.1. Chemicals

3-Fluorobenzoate was obtained from Fluka, 2- and

4-fluorobenzoate were purchased from Fluorochem(Derbyshire, UK) and 3-fluorcatechol was acquired

from Acros Organics (NJ, USA). Fluoro-substituted

3,5-cyclohexadiene-1,2-diol-1-carboxylic acids (dihydro-

dihydroxybenzoates) were obtained from Alcaligenes

eutrophus B9 [12], which was cultured on a fructose

mineral medium containing 3-fluorobenzoate (1 mM).

The A. eutrophus strain was a gift from Andrew Myers

(Department of Chemistry and Chemical Biology, Har-vard University, USA).

2.2. Isolation and characterisation of a 3-fluorobenzoate

degrading bacterium

Soil from Co. Wexford, Ireland that had been previ-

ously treated with a fluorinated herbicide (Punch C) was

used to inoculate E2 mineral medium [13] containing 5mM 3-fluorobenzoate as the sole source of carbon. After

14 days incubation at 25 �C in 250 ml Erlenmeyer flasks

on an orbital shaker, the fluoride ion concentration was

measured using an ion-selective electrode (Orion Model

96.06) using the method of Cooke [14]. Agar plates con-

taining E2 medium and 3-fluorobenzoate were inoculat-

ed with 0.1 ml of the suspension and one of the colonies

(strain HB-1) was purified by streaking on the same agarmedium. Purity was assessed on nutrient agar plates and

by light microscopy (Gram staining).

The 16S rRNA gene was amplified by PCR with

Taq polymerase (Promega) as recommended by the

manufacturer using the oligonucleotides F27 (5 0-AGA-

GTTTGATCMTGGCTCAG-3 0) and R1492 (5 0-TAC-

GGYTACCTTGTTACGACTT-3 0). The reaction

mixture was incubated at 94 �C for 2 min and was sub-sequently subjected to 25 cycles of 94 �C for 45 s, 52 �Cfor 45 s, 74 �C for 2 min, followed by incubation at 74

�C for 7 min.

Dideoxy sequencing reactions were done with the

CEQ DCTS Kit as described by the manufacturer

(Beckman) using oligonucleotides complementary to

conserved regions in the 16S rRNA gene. The nucleotide

sequence was determined using a Beckman CEQ 2000automatic sequencer; nucleotide sequence data were

compiled using the Staden package [15].

2.3. Identification of intermediates

The isolated bacterium was grown in Erlenmeyer

flasks containing E2 medium (50 ml) supplemented with

fluorobenzoate (2.5 or 5 mM) incubated on an orbitalshaker at 25 �C. Portions of the culture were removed

and centrifuged at various intervals and centrifuged,

and the supernatants stored frozen.

The supernatants were analysed by 19F nuclear mag-

netic resonance spectroscopy (19F NMR) using a Bruker

Avance 500 MHz spectrometer. Supernatants (0.5 ml)

were transferred to NMR tubes and D2O (0.2 ml) was

added to provide a lock signal. Identification of com-pounds present in the supernatants was also determined

by gas chromatography–mass spectrometry (GC–MS)

using a Hewlett–Packard 6890 gas chromatograph

linked to a 5973 mass selective detector controlled by

Chemstation software. Culture supernatants (200 ll or1 ml) were freeze-dried and either resuspended in chloro-

form or derivatised by adding N-methyl-N-(trimethylsi-

lyl) trifluoroacetamide (MSTFA, 200 ll) and heating for1 h at 100 �C. Samples in chloroform (1 ll) were injectedin the splitless mode onto a Hewlett–Packard Ultra 1

column (12 m·0.2 mm·0.33 lm) and the oven temper-

ature held at 50 �C for 1 min then raised to 280 �C at 10

�Cmin�1. The derivatised samples (1 ll) were injected

onto a Chrompak CP-Sil 19 CB column (25 m·0.25mm·0.2 lm) employing a split ratio of 10:1 and the

oven temperature held at 120 �C for 1 min then raisedto 270 �C at 10 �Cmin�1.

3. Results

3.1. Isolation and characterisation of strain HB-1

Degradation of 3-fluorobenzoate in the soil enrich-ment cultures was determined by measuring the concen-

tration of free fluoride ion. Cultures containing elevated

F.G. Hidde Boersma et al. / FEMS Microbiology Letters 237 (2004) 355–361 357

concentrations of fluoride (>70% defluorination) were

visibly darker than those in which no free fluoride was

detected, probably as a consequence of fluorosubstituted

catechol accumulation. Upon further culturing on agar

supplemented with 3-fluorobenzoate one colony type

was predominant, which was subsequently isolated. Mi-croscopic analysis determined this bacterium to be a

small Gram negative rod. Sequence comparison of the

16S rRNA gene with sequences in the GenBank data-

base revealed that the organism was a sphingomonad,

which was very closely related to Sphingomonas yan-

oikuyae (100% sequence identity with 918 nucleotides

of 16S rRNA gene). The sequence has been deposited

in GenBank under the Accession No. AY387397.

3.2. 19F NMR analysis of supernatants

Culture supernatants from various stages of growth

were analysed by 19F NMR spectroscopy to monitor

the catabolism of 3-fluorobenzoate and the spectra ob-

tained from this analysis are shown in Fig. 1(a). As

-113.00 -112.80

-114 -112 -110

-120-114-108-102

COOH

F

(a)

(b)

Fig. 1. (a) Decoupled 19F NMR spectra of culture supernatant of Sphingomo

The spectra were not referenced to CFCl3, thus the chemical shift values are s

NMR spectrum of Alcaligenes eutrophus B9 culture supernatant grown in t

coupled signals.

growth proceeded 3-fluorobenzoate (�113.9 ppm) disap-

peared and several new signals appeared, indicating the

accumulation of fluorinated metabolites in the culture

fluid. By day 4 the strongest signal was that of fluoride

ion (broad singlet,�119.5 ppm), thus most of the 3-fluor-

obenzoate was completely mineralised. The signal at�107.7 ppm observed at day 2 is a doublet (J=21.6 Hz)

and has a similar chemical shift and coupling constant

to that of 2-fluoromuconate [16]. Comparisonwith an au-

thentic standard identified the very small signal at�136.2

ppm as 3-fluorocatechol and GC–MS analysis of the su-

pernatant confirmed the presence of this compound. It

was suspected that the remaining two signals at �112.9

and �118.7 ppm were 3- and 5-fluoro-1,2-dihydro-1,2-dihydroxybenzoates. No authentic standard of either

compound was available and there are no 19F NMR data

in the literature. However, biotransformation of 3-fluor-

obenzoate byA. eutrophus B9 yields fluorosubstituted di-

hydrodihydroxybenzoates [12], as this bacterium has a

defective dehydrogenase responsible for the conver-

sion of dihydrodihydroxybenzoate to catechol. The

-118.80-118.60

ppm-118 -116

-138 -132-126 ppm

F-

Day 0

Day 2

Day 4

nas sp. HB-1 at various stages throughout growth on 3-fluorobenzoate.

hifted by approximately 4 ppm with respect to literature values. (b) 19F

he presence of 3-fluorobenzoate. The insets are expansions of the 1H-

-140-130-120-110-100-90-80 ppm

Day 2

Day 6

COOH

F

COOHCOOH

F

Fig. 2. Decoupled 19F NMR spectrum of culture supernatant of Sphingomonas sp. HB-1 grown on 2-fluorobenzoate. The metabolite at �107.7 ppm

is a doublet (J=21.6 Hz).

358 F.G. Hidde Boersma et al. / FEMS Microbiology Letters 237 (2004) 355–361

19F NMR spectrum of the culture supernatant from A.

eutrophus B9 grown in the presence of 3-fluorobenzoate

is shown in Fig. 1(b). It could be demonstrated by analy-

sing a mixture of the culture supernatants from both or-ganisms that the unidentified signals present in the

Sphingomonas sp. supernatant had identical chemical

shifts to the signals observed in the A. eutrophus superna-

tant. The coupling constants of both signals are very sim-

ilar indicating coupling of fluorine to hydrogen atoms on

the adjacent carbon (3JF–H=12 Hz) and to hydrogen at-

oms that are two carbons removed (4JF–H=6 Hz), but

the splitting patterns are different (Fig. 1(b)). The signalat �112.9 ppm (doublet of doublets) is probably that of

3-fluorodihydrodihydroxybenzoate, where the fluorine

is coupled to the hydrogen at C4 yielding a doublet, which

is further split by coupling to the hydrogen at either C5

or C2. The signal at �118.7 ppm is thus 5-fluor-

odihydrodihydroxybenzoate with fluorine coupling to

equivalent hydrogen atoms at C4 and C6 yielding a trip-

let, and to the hydrogen at C3, which further splits the sig-nal. Moreover, GC–MS analysis of culture supernatants

from Sphingomonas sp. HB-1 revealed two compounds

with almost identical mass spectra and molecular ions

that corresponded to the calculated mass of silylated flu-

orosubstituted dihydrodihydroxybenzoates; compounds

with the same retention times and mass spectra were de-

tected in A. eutrophus culture supernatant.

When E2 medium containing 2- or 4-fluorobenzoateas sole carbon and energy sources was inoculated with

Sphingomonas sp. HB-1 (grown on 3-fluorobenzoate),

growth was visible after 4–6 days incubation. Analysis

of the supernatants revealed that 2-fluoromuconate

and fluoride accumulated in the 2-fluorobenzoate-grown

culture (Fig. 2). Fluoride accumulated in the 4-fluor-

obenzoate-grown culture along with a small amount of

another compound at �116.4 ppm (Fig. 3), which wasnot readily identifiable from data in the literature. The

splitting pattern and coupling constants indicated that

it might be 4-fluorodihydrodihydroxybenzoate: fluorine

coupling to equivalent hydrogens at C3 and C5 yielding

a triplet (J=11.9 Hz) and longer range coupling to the

hydrogen at C6 or C2 resulting in further splitting(J=6 Hz). GC–MS analysis of the derivatised superna-

tant detected the presence of a fluorosubstituted

dihydrodihydroxybenzoate. Interestingly, no 2- and 3-

fluorobenzoate remained in the supernatants after 4

days incubation, but cultures grown on 4-fluorobenzo-

ate took much longer (8 days) to fully catabolise the

substrate. This was consistent with the appearance of

turbidity in the culture flasks.

4. Discussion

A catabolic pathway for the degradation of 3-fluor-

obenzoate by Pseudomonas sp. B13 was proposed by

Schreiber et al. [10] and is shown in Fig. 4. When grown

on 3-chlorobenzoate this strain could co-metabolise 3-fluorobenzoate; 2-fluoromuconic and 3- and 4-fluoro-

catechol were detected in the culture medium. However,

the organism could not use 3-fluorobenzoate as a sole

carbon and energy source. Engesser et al. [11] described

the isolation of a bacterial strain, FLB 300, which could

use 3-fluorobenzoate as a sole source of carbon and en-

ergy. Fluoride and 2-fluoromuconic acid were detected

in the culture fluid, and it was suggested that the samepathway as that proposed by Schreiber et al. (Fig. 4)

was responsible for the catabolism of the 3-fluorobenzo-

ate in this strain. However, it was also concluded that on

the basis of the ratio of fluoride and 2-fluoromuconate,

the predominant pathway was via initial 1,6-dioxygen-

ation, yielding 4-fluorocatechol. It was further suggested

that the regioselectivity of the initial dioxygenation was

the critical step in determining whether or not an organ-ism could use 3-fluorobenzoate as a sole source of car-

bon and energy, for example, the initial dioxygenation

-140 -130 -120 -110 -100 -90 ppm

Day 0

Day 6

Day 8

OHOH

H

HOOC

F

COOH

F

-116.40 -116.30

Fig. 3. 19F NMR spectra of culture supernatants of Sphingomonas sp. HB-1 grown on 4-fluorobenzoate. The inset is an expansion of the 1H-coupled

signal at �116.4 ppm.

F.G. Hidde Boersma et al. / FEMS Microbiology Letters 237 (2004) 355–361 359

in Pseudomonas sp. B13 is in the 1,2-position, thus toxic

3-fluorocatechol accumulates [17].

Our studies have succeeded in isolating a microor-

ganism that can use 3-fluorobenzoate as a sole sourceof carbon and energy. Comparison of the 16S rRNA

gene sequence indicated that the organism is a sphin-

gomonad. Members of this genus are known to utilise

xenobiotic compounds, including pentachlorophenol

and c-hexachlorocyclohexane [18,19], but no studies

with fluorobenzoates have been reported thus far.

The culture supernatant of Sphingomonas sp. HB-1

was analysed throughout batch culture growth on3-fluorobenzoate using 19F NMR and GC–MS tech-

niques, and several fluorinated compounds were de-

tected. The most prominent signal observed by 19F

NMR was that of fluoride ion, suggesting that most

of the 3-fluorobenzoate was completely mineralised

by this organism. Signals were also present on the19F NMR spectra that had chemical shifts and cou-

pling constants similar to those of 3-fluorocatechol,2-fluoromuconic acid and 3- and 5-fluoro-1,2-dihy-

dro-1,2-dihydroxybenzoate. The presence of the

fluorosubstituted 1,2-dihydro-1,2-dihydroxybenzoates

was confirmed by GC–MS analysis. Thus, it is most

likely that this organism employs the same pathway

to catabolise 3-fluorobenzoate as shown in Fig. 4

and since fluoride was the major product, the initial

dioxygenation must favour the 1,6 positions, as sug-gested by Engesser et al. Furthermore, the presence

of 2-fluoromuconic acid in the supernatant may also

indicate that this strain has a catechol 1,2-dioxygenase

that will utilise 3-fluorocatechol as a substrate, but

further enzymatic studies are necessary to confirm

this. 4-Fluorocatechol and 3-fluoromuconic acid were

not detected by either 19F NMR or GC–MS analysesof culture supernatants. Presumably these intermedi-

ates were readily transformed by the enzymes catechol

1,2-dioxygenase and muconate cycloisomerase, and

hence did not accumulate in the culture fluid.

Not unexpectedly, Sphingomonas sp. HB-1 can also

utilise the 2- and 4-fluoro isomers as growth substrates.

In addition to fluoride, 2-fluoromuconic acid accumu-

lates in cultures grown on 2-fluorobenzoate, consistentwith previous observations in other bacteria, and in cul-

tures grown on 4-fluorobenzoate, a fluoro-substituted

dihydrodihydroxybenzoate was detected. It is most

likely that this is the 4-fluoro isomer, since it had a dif-

ferent chemical shift and splitting pattern to the 3- and

5-fluoro isomers observed in 3-fluorobenzoate grown

cells, and previous investigations with 4-fluorobenzo-

ate-degrading bacteria indicate that the initial productof dioxygenase attack is 4-fluoro-1,2-dihydro-1,2-di-

hydroxybenzoate.

This study provides confirmatory evidence, in the

form of 19F NMR spectral analyses, that the ability of

an organism to utilise 3-fluorobenzoate as a growth sub-

strate is determined by the regiospecificity of the benzo-

ate dioxygenase as proposed by Engesser et al. [11].

While this organism was isolated from a soil that wascontaminated with a fluorinated compound, it is not

known whether this is a significant factor in the ability

of Sphingomonas sp. HB-1 to degrade 3-fluorobenzoate.

COOH

F

F

OHOH

H

HOOC OHOH

H

F

HOOC

OH

OH

F

OH

F

OH

COOHCOOH

F

COOHCOOH

F

COOHCOOH

O

HF

3-fluorobenzoic acid

fluorinated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acids(fluoro-1,2-dihydro-1,2-dihydroxybenzoic acids)

fluorocatechols

fluoromuconic acids

oxoadipic acid

Fig. 4. The pathway of 3-fluorobenzoate biodegradation [10].

360 F.G. Hidde Boersma et al. / FEMS Microbiology Letters 237 (2004) 355–361

Acknowledgement

The authors thank Marion Carolan for culturing Al-

caligenes eutrophus and Evelyn Doyle, Wim Meijer and

Kevin O�Connor for helpful discussions.

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