APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2010, p. 3590–3598
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 11
Growth of Bacteria on 3-Nitropropionic Acid as a Sole Source of
Carbon, Nitrogen, and Energy?
Shirley F. Nishino, Kwanghee A. Shin, Rayford B. Payne,† and Jim C. Spain*
School of Civil and Environmental Engineering, Georgia Institute of Technology, 311 Ferst Dr., Atlanta, Georgia 30332-0512
Received 1 February 2010/Accepted 2 April 2010
3-Nitropropionic acid (3NPA) is a widespread nitroaliphatic toxin found in a variety of legumes and fungi.
Several enzymes have been reported that can transform the compound, but none led to the mineralization of
3NPA. We report here the isolation of bacteria that grow on 3NPA and its anion, propionate-3-nitronate (P3N),
as the sole source of carbon, nitrogen, and energy. Experiments with resting cells, cell extracts, and purified
enzymes indicate that the pathway involves conversion of 3NPA to P3N, which upon denitration yields malonic
semialdehyde, nitrate, nitrite, and traces of H2O2. Malonic semialdehyde is decarboxylated to acetyl coenzyme
A. The gene that encodes the enzyme responsible for the denitration of P3N was cloned and expressed, and the
enzyme was purified. Stoichiometry of the reaction indicates that the enzyme is a monooxygenase. The gene
sequence is related to a large group of genes annotated as 2-nitropropane dioxygenases, but the P3N mono-
oxygenase and closely related enzymes form a cluster within COG2070 that differs from previously character-
ized 2-nitropropane dioxygenases by their substrate specificities and reaction products. The results suggest
that the P3N monooxygenases enable bacteria to exploit 3NPA in natural habitats as a growth substrate.
Large-scale release of synthetic nitroaromatic compounds to
the biosphere followed the invention of nitrobenzene around
1830. In less than 200 years, microorganisms adapted to the
presence of nitroaromatic compounds in the environment by
developing catalytic pathways to exploit them as growth sub-
strates. Such rapid development suggests that the pathways did
not develop de novo but evolved from preexisting degradation
pathways such as might be found in microorganisms that de-
grade naturally occurring compounds.
3-Nitropropionic acid (3NPA) is a widespread naturally oc-
curring nitroaliphatic compound. It is a principal toxic compo-
nent of Astragalus locoweeds and has been found in hundreds
of species of legumes (20, 39) and a variety of fungi (6). The
compound causes irreversible inhibition of succinate dehydro-
genase, which makes it deadly to eukaryotes (1). Plants that
make 3NPA also contain an enzyme, 3NPA oxidase (NPAO)
(19, 20), which converts the compound to malonic semialde-
hyde (MSA) to protect the plant against the toxic effects of the
compound (20). Given its widespread occurrence, we hypoth-
esized that there must be bacteria in soil that degrade 3NPA
and play a major role in determining the flux of the compound.
Although bacteria that degrade 3NPA have previously been
sought, the focus has been on organisms that ingest 3NPA-
containing plant matter. Rumen microorganisms reduce 3NPA
to ?-alanine (4), and in the grasshopper gut, 3NPA is bound to
glycine to form inert conjugates which are then eliminated
(24). The plant enzyme NPAO converts 3NPA and O2to MSA,
nitrate, nitrite, and hydrogen peroxide (19). It is similar to
propionate-3-nitronate (P3N) oxidase (P3NO; EC 126.96.36.199)
from Penicillium atrovenetum that converts the P3N form of the
compound to MSA (36). The enzymes mentioned above are
“orphan enzymes” (28), which means that the gene(s) has not
been identified. None of the previously studied microorgan-
isms can use 3NPA as a growth substrate, and the physiological
roles of the enzymes have not been established.
MSA appears to be a central intermediate in the eukaryotic
transformation of 3NPA and its analogs. However, the trans-
formations involve distinctly different types of reactions and
metabolites released. P3N and 3NPA release nitrate and ni-
trite in a 2:1 ratio when attacked by the fungal or plant oxi-
dases. When 3NPA is reduced to ?-alanine by rumen micro-
organisms, ?-alanine is further metabolized (4), possibly by
deamination to MSA (18).
We report here the isolation from soil of aerobic bacteria
that grow on 3NPA as the sole source of carbon, nitrogen, and
energy. The genes that encode the initial enzymes of the deg-
radation pathway were cloned, and recombinant proteins were
purified and partially characterized to allow determination of
the initial steps in the catabolic pathway.
(Preliminary reports of this work have been presented pre-
viously at the 106th General Meeting of the American Society
for Microbiology [32a] and the 108th General Meeting of the
American Society for Microbiology [32b].)
MATERIALS AND METHODS
Isolation and growth of bacteria. Bacteria were isolated by selective enrich-
ment in nitrogen-free minimal medium (BLK) (5) containing 3NPA (100 ?M to
1 mM). After several transfers, the cultures were spread onto agar plates
containing BLK with 3NPA (500 ?M) and incubated at 30°C until colonies
appeared. Cultures that grew on 3NPA plates were selected for identification
and further study. 16S rRNA gene analysis was performed by MIDI Labs
Large cultures were grown in 1 liter of BLK containing twice the normal
concentration of MgSO4plus 3NPA (1 mM). When the 3NPA disappeared from
the medium, it was added twice more to a final concentration of 1 mM. Some
cultures were grown on 3NPA (500 ?M) as the nitrogen source with succinate (7
* Corresponding author. Mailing address: School of Civil and Envi-
ronmental Engineering, Georgia Institute of Technology, 311 Ferst
Dr., Atlanta, GA 30332-0512. Phone: (404) 894-0628. Fax: (404) 894-
8266. E-mail: email@example.com.
† Present address: Center of Marine Biotechnology, University of
Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore,
?Published ahead of print on 9 April 2010.
mM) or acetate (10 mM) as the carbon source. Cultures were harvested by
centrifugation, washed twice with phosphate buffer (20 mM; pH 7.0), and stored
on ice until used in assays. Cells were broken by three passages through a French
pressure cell followed by centrifugation at either 26,000 ? g or 100,000 ? g. Cell
extracts were stored on ice until used.
Enzyme assays. 3NPA denitrase was assayed by measurement of oxygen up-
take in the presence of 3NPA or P3N. Reaction mixtures consisted of cell
extracts or purified protein (1 to 500 ?g protein) in phosphate (20 mM; pH 7.8)
or Tris buffer (100 mM; pH 7.8). The reaction was started by addition of
substrate. MSA formed from P3N was quantified spectrophotometrically by
following the NADH-dependent disappearance of MSA catalyzed by ?-hydroxy-
butyrate dehydrogenase (BDH) (36). Malonyl coenzyme A (malonyl-CoA) re-
ductase was measured in the forward direction by following the disappearance of
NADPH at 365 nm (22) and in the reverse direction by following the reduction
of NADP (22). MSA decarboxylase was assayed by following the disappearance
of MSA in the absence of cofactors (17). MSA oxidative decarboxylase was
measured by following the CoA-dependent reduction of NADP to NADPH in
the presence of MSA (29). Acetaldehyde dehydrogenase was measured by fol-
lowing the conversion of NAD to NADH in the presence of acetaldehyde (17).
Library construction. DNA (approximately 5 mg) was extracted from cultures
of Pseudomonas sp. strain JS189 with the Promega SV genomic DNA purifica-
tion system (Madison, WI) according to the manufacturer’s instructions. The
resulting DNA was sheared by vortexing. DNA fragments approximately 30 kb in
size were gel purified, ligated into the fosmid vector pCC1Fos, packaged into
phage, and transfected into Escherichia coli strain EPI300-T1 according to the
manufacturer’s directions (CopyControl fosmid library production kit; Epicentre
Biotechnologies, Madison, WI) to create a library of several thousand recombi-
nant E. coli clones.
Screening for P3N monooxygenase activity (pnoA). Clones were transferred to
96-well plates containing tryptic soy broth supplemented with NH4Cl (1 mM),
chloramphenicol (20 ?g/ml), and 1? concentrated fosmid induction solution
(Epicentre Biotechnologies, Madison, WI) and grown for 16 h at 30°C. After
growth, the library was replica plated onto BLK supplemented with NH4Cl (1
mM), chloramphenicol (20 ?g/ml), 1? concentrated fosmid induction solution,
P3N (200 ?M), and sodium succinate (200 ?M) and then incubated for 12 to 16 h
at 30°C. Clones were screened for nitrite release from P3N.
The location of pnoA on the fosmid was determined by transposon mutagen-
esis followed by a second round of screening for loss-of-function mutants. The
fosmid bearing pnoA was purified using the FosmidMax DNA purification kit
(Epicentre Biotechnologies, Madison, WI) and then randomly mutated in vitro
with the EZ-Tn5 ?KAN-2? insertion kit (Epicentre Biotechnologies, Madison,
WI). The fosmid::Ez-Tn5 ?KAN-2? fusion was reintroduced into E. coli strain
EPI300-T1 by electroporation. Mutants in which the transposon had inserted
into the fosmid were selected by growth on LB media supplemented with chlor-
amphenicol (20 ?g/ml) and kanamycin (25 ?g/ml). Kanamycin-resistant trans-
poson mutants were screened for the ability to release nitrite from P3N. Loss-
of-function mutants were sequenced using the KANREV primer (41) to
determine the insertion site of the transposon. The acquired sequence was used
to manually design additional primers for subsequent rounds of primer walking.
Approximately 10 kb of sequence was obtained and assembled into a 2-kb contig
with CAP3 (21). Open reading frames were determined by ORFFinder (http:
Cloning and expression of closely related genes. The Basic Local Alignment
Search Tool (BLAST) (3) was used to perform a homology search of the amino
acid sequence of PnoA against the protein database in GenBank. Several of the
most similar genes (Table 1) were amplified by PCR. The reaction mixture (20
?l) contained genomic DNA (68 to 115 ng), primers (0.25 ?M each), de-
oxynucleoside triphosphates (dNTPs; 0.4 mM each), buffer (1?), and Promega
GoTaq hot-start polymerase (2 U). Amplifications (30 cycles) were carried out as
follows: 95°C for 1 min, 54°C for 30 s, and 72°C for 1 min, after initial denatur-
ation at 95°C for 10 min. The PCR products were cut with BamHI and XhoI and
cloned into the pET-21a vector (Novagen, Gibbstown, NJ). The resulting con-
structs were transformed into E. coli Top10 (Invitrogen, Carlsbad, CA) for
plasmid propagation and maintenance. The constructs were transformed into E.
coli BL21 Star (DE3) (Invitrogen) for expression. Cells were grown in 30 ml of
LB medium containing Overnight Express autoinduction system I (Novagen) at
37°C. When the optical density at 600 nm (OD600) reached 0.8 to 0.9, the
temperature was reduced to 25°C, and the cultures were further incubated for
10 h. Cells were harvested by centrifugation, washed twice with phosphate buffer
(20 mM; pH 7.5) and stored on ice until used. Trees and alignments were
constructed with Geneious Pro 4.7.4.
Protein purification. His tag fusions were constructed using the pBAD102-D/
TOPO expression system (Invitrogen). Genes of interest were cloned in frame
TABLE 1. Putative P3N monooxygenases
E. coli strain
Size of protein
Pseudomonas sp. JS189
Burkholderia phytofirmans PsJN
Burkholderia phytofirmans PsJN
Pseudomonas aeruginosa PAO1
aRestriction sites are underlined.
VOL. 76, 2010 BIODEGRADATION OF 3-NITROPROPIONIC ACID3591
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3598 NISHINO ET AL.APPL. ENVIRON. MICROBIOL.