Role of IncP-1? Plasmids pWDL7::rfp and pNB8c in Chloroaniline
Catabolism as Determined by Genomic and Functional Analyses
J. E. Król,a* J. T. Penrod,bH. McCaslin,c* L. M. Rogers,aH. Yano,aA. D. Stancik,aW. Dejonghe,d* C. J. Brown,aR. E. Parales,b
S. Wuertz,c,eand E. M. Topa
Department of Biological Sciences, Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, Idaho, USAa; Department of Microbiology,
University of California, Davis, Davis, California, USAb; Department of Civil and Environmental Engineering, University of California, Davis, Davis, California, USAc; Laboratory
of Microbial Ecology and Technology, Ghent University, Ghent, Belgiumd; and Singapore Centre on Environmental Life Sciences Engineering, School of Biological
Sciences, and School of Civil and Environmental Engineering, Nanyang Technological University, Singaporee
detectedoneitherplasmid,suggestingthatonlytheupper3-CAdegradationpathwaywaspresent.The dcaA1A2B geneproducts
expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli. Slight differ-
azo dyes, and polyurethanes, and they also accumulate in the en-
vironment as a result of the microbial degradation of herbicides
(11). Anilines, especially chloroanilines, are toxic and carcino-
genic. There are reports of a few bacterial strains that are capable
of degrading mono- and dichloroanilines (35, 64); however, the
bacteria (57). The general pathway for chloroaniline degradation
is thought to follow the typical aerobic biodegradation pathway
for chlorinated aromatic compounds. First, a peripheral (upper)
pathway generates chlorocatechol by oxidative deamination.
Then, modified ortho- or meta-cleavage (lower) pathways con-
vert chlorocatechol to tricarboxylic acid (TCA) cycle intermedi-
ates (22, 25, 48). The accumulation of upper-pathway intermedi-
involved in chloroaniline degradation have been found on plas-
mids as well as on bacterial chromosomes (7, 8, 14, 26).
Plasmid-mediated horizontal transfer of catabolic genes con-
tributes to the ability of bacterial communities to degrade toxic
man-made compounds, and this natural process can be exploited
in bioaugmentation approaches (60, 56). While we have gained
catabolic plasmids and their role in biodegradation of xenobiotic
compounds, relatively few such plasmids have been rigorously
analyzed and compared. In order to understand the evolution of
these mobile elements and their contribution to the removal of
toxic compounds in our environment, complete plasmid se-
significance of their gene products.
niline and its derivatives are used industrially in the produc-
Several bacterial strains isolated for their ability to degrade
man-made, chlorinated organic compounds carry genes en-
coding the relevant degradation pathways on plasmids of the
incompatibility group IncP-1, which transfer to and replicate
in a broad range of hosts (55). A few of these plasmids have
recently been completely sequenced: pJP4, pEST4011 [2,4-
dichlorophenoxyacetic acid degradation (58, 61)], pUO1 [ha-
loacetate degradation (44)], pADP-1 [atrazine degradation
(33)], pA81 [chlorobenzoate degradation (23)], and pCNB1 [4-
chloronitrobenzene degradation (32)]; details regarding most of
these plasmid sequences are also summarized in a recent review
(60). While several catabolic plasmids that encode (partial)
(chloro)aniline degradation pathways have been described, such
as pCIT1 (2, 34), pTDN1 (18, 40), pYA1 (17), pNB1, pNB2,
pNB8c, pC1 (7), and pWDL7 and pTB30 (14), their complete
genome sequences have not yet been reported. Some of these
plasmids wereshownto transfer
3-chloroaniline (3-CA) as the sole nitrogen or occasionally the
the ability touse
Received 7 November 2011 Accepted 12 November 2011
Published ahead of print 18 November 2011
Address correspondence to E. M. Top, firstname.lastname@example.org.
*Present address: J. E. Król, Department of Microbiology and Cell Science,
University of Florida, Gainesville, Florida, USA; H. McCaslin, Larry Walker Associates,
Davis, California, USA; W. Dejonghe, Separation and Conversion Technology,
Flemish Institute for Technological Research, Mol, Belgium.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
aem.asm.org 0099-2240/12/$12.00 Applied and Environmental Microbiologyp. 828–838
clear if all of the genes necessary for complete degradation are
located on the plasmids.
In this study, we describe the complete genome sequences of
plasmids involved in 3-CA and aniline degradation: pWDL7::rfp,
teroni strain WDL7, and pNB8c, a 60.4-kb plasmid from Delftia
acidovorans strain B8c. These strains were obtained from an or-
chard soil with a 10-year history of treatment with a mixture of
linuron, diuron, and simazine, and from activated sludge of a
plasmid pWDL7, C. testosteroni WDL7 and Cupriavidus pinatu-
bonensis JMP228n (formerly Cupriavidus necator), were shown to
completely degrade 3-CA, suggesting that the plasmid encoded
pathways for at least partial and possibly complete degradation of
this recalcitrant and toxic compound (13, 14). In contrast, plas-
mid pNB8c conferred aniline but not chloroaniline transforma-
tion in C. pinatubonensis JMP228gfp (7). Based on sequence and
hybridization results, both plasmids had been assigned to the ?
subgroup of the IncP-1 plasmids (7, 14).
Our specific objectives were to (i) determine, analyze, and
compare the complete sequences of a derivative of plasmid
pWDL7 labeled with the red fluorescent protein, designated
pWDL7::rfp, and its close relative, plasmid pNB8c, and infer their
roaniline catabolic genes and determine their transcriptional ac-
tivity and function; and (iii) determine the utility of pWDL7::rfp
as a tool to augment the catabolic capabilities of activated sludge
exposed to 3-CA. The motivation for sequencing the rfp-marked
version of pWDL7 and not the wild type is its utility in bioaug-
mentation studies due to the presence of fluorescent and antibi-
otic resistance markers.
MATERIALS AND METHODS
listed in Table 1. Activated sludge used in batch mating studies was ob-
tained from the University of California, Davis, wastewater treatment
plant (Davis, CA).
medium ) with appropriate antibiotics at 30°C or 37°C. In experi-
minimal medium without additional carbon (MMO) was used (47). In
MMO, 3-CA was the sole carbon source and ammonium (2.5 mM
(NH4)2SO4) was available as a nitrogen source. In experiments to deter-
mine if 3-CA was used as a nitrogen source, minimal medium without
nitrogen (MMN) was used, in the presence or absence of an additional
carbon source such as pyruvate (7). The media were prepared as previ-
ously described (14).
tein gene (rfp) and kanamycin resistance. The plasmid pWDL7 was
tagged with the mini-Tn5 transposon from plasmid pTTN151 (52) as
previously described (54), and the resulting plasmid was designated
pWDL7::rfp. Tagged plasmids were transferred to C. pinatubonensis
TABLE 1 Characteristics of strains and plasmids
Strain or plasmidCharacteristicsa
E. coli K12Nal
E. coli DH5?
MG1655 (ATCC 47076), F???ilvG rfb-50 rph-1 Nalr
F??80dlacZ?M15 ?(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK?mK?) phoA supE44 ??
thi-1 gyrA96 relA1
?(ara-leu) araD ?lacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA ?pir phage lysogen
F?mcrB mrr hsdS20(rB?mB?) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(Smr)
Isolate from linuron-degrading consortium; 3-CACN,3,4-DCACN; original host of plasmid pWDL7
Isolate from a wastewater treatment plant; 3-CACN; original host of plasmid pNB8c
Reference strain used as recipient
Reference strain used as recipient
Rifr; plasmid host strain in dca promoter studies
Nalrderivative of JMP228; plasmid host strain in mini-Tn5 tagging protocol
E. coli CC118 ?pir
E. coli HB101
C. testosteroni WDL7
D. acidovorans B8c
C. testosteroni ATCC 11996
D. acidovorans ATCC 15668
C. pinatubonensis JMP228
C. pinatubonensis JMP228n
P. putida UWC3
P. putida UWC3(pWDL7::rfp)
IncP-1? KmrCmrRFP; encodes chloroaniline dioxygenase
IncP-1?; encodes chloroaniline dioxygenase
R6Kori AmprKmrCmrRFP; transposon delivery vector
pGem3Zf? with pT7-gfpmut3 in SmaI site; reporter plasmid; Ampr
pG-GFP with pWDL7 pdca promoter
pG-GFP with pNB8c pdca promoter
pBBR1MCS-2 carrying pWDL7::rfp dcaA1A2B genes cloned into XhoI/ClaI sites; Kmr
pUC18 with HindIII/KpnI fragment of pJTP500 plac-dcaA1A2B; Ampr
pJPC13 with EcoRI/HindIII fragment carrying pWDL7::rfp dcaQT; Cmr
pJPC13 with EcoRI/HindIII fragment carrying pNB8c dcaQT; Cmr
aNalr, nalidixic acid resistance, Rifr, rifampin resistance; Kmr, kanamycin resistance; Cmr, chloramphenicol resistance; Ampr, ampicillin resistance; ilv, isoleucine, leucine, and
valine auxotrophy gene; RFP, red fluorescent protein; ATCC, American Type Culture Collection. Superscript “N” and “C” indicate use of the indicated compound as the sole
nitrogen and carbon source, respectively.
Plasmid-Mediated Chloroaniline Degradation
February 2012 Volume 78 Number 3 aem.asm.org 829
mid pWDL7::rfp was then transferred in a plate mating (see below) from
followed by selection on LB medium with rifampin (Rif; 50 mg/liter) and
Km (50 mg/liter).
Plasmid DNA isolation, cloning, restriction analysis, sequence de-
termination, assembly, and annotation. To obtain sufficient plasmid
DNA of high quality for sequence determination by shotgun cloning,
plasmids were first transferred by conjugation from JMP228n(pWDL7::
rfp) and B8c to E. coli K12Nal, a nalidixic acid-resistant mutant of
MG1655. In the case of pNB8c, transconjugants were not selected for a
specific phenotype but were found by randomly choosing recipient colo-
jugants were minimally processed to avoid mutations in the plasmids.
Plasmid DNA to be used for sequence determination and additional ana-
CA) according to the manufacturer’s instructions for low-copy-number
plasmids. Standard methods were used for restriction analysis and pulse
modification enzymes were purchased from New England BioLabs (Ips-
wich, MA) and Fermentas Inc. (Glen Burnie, MD). DNA fragments were
purified with a QIAquick gel extraction kit (Qiagen).
The genome sequences of plasmids pWDL7::rfp and pNB8c were de-
termined by shotgun sequencing at the DOE Joint Genome Institute
(Walnut Creek, CA), with 10? coverage for each plasmid. Regions of
poor sequence quality were resequenced at the University of Idaho by
PCR amplification followed by sequencing using a BigDye Terminator
v.3.1 cycle sequencing kit and a 3730 DNA analyzer (Applied Biosystems,
Carlsbad, CA). The sequence was automatically annotated by the J. Craig
Venter Institute Annotation Service (http://www.jcvi.org/annotation
/service/) and further annotated manually using Manatee (manatee
Bioinformatics analyses and software. Similarity searches were per-
this work were generated using GENtle software (Magnus Manske, Uni-
versity of Cologne, Germany). Plasmid alignments were performed using
Mauve (12) and then refined and rendered graphically using TRAPPIST,
a Python-based sequence map and alignment drawing tool for plasmids
(G. Van der Auwera, unpublished data), with identity scoring by Clust-
alW. The pdca promoter region was analyzed using BPROM (Softberry
Inc., Mount Kisco, NY).
deduced amino acid sequences of 24 genes from the 26 plasmids were
aligned individually using MUSCLE (15) and then concatenated into a
single alignment. RAxML-VI-HPC was used to infer a maximum likeli-
hood phylogeny for the aligned sequences (46). The phylogeny with the
largest likelihood was chosen from 100 iterations that altered the starting
tree. The phylogeny inferred by this method was not substantially differ-
Proteins used were KfrA, KfrB, KlcA, KleE, KorA, KorB, KorC, TraD,
TrbG, TrbI, TrbJ, and TrbK. The plasmids used in the analysis (and their
accession numbers) were pR751 (NC_001735), pADP1 (NC_004956),
pB4 (NC_003430), pA81 (NC_006830), pUO1 (NC_005088), pB10
(NC_004840), RK2 (NC_001621), pB3 (NC_006388), pA1 (NC_007353),
pCNB1 (NC_010935), pJP4 (NC_005912), pAKD4 (GQ983559), pSP21
(NC_007680), pAOVO02 (NC_008766), pTB11 (NC_006352), pB8
(NC_007502), pB11 (CP002152), pB5 (CP002151), pKJK5 (NC_008272),
QKH54 (NC_008055), pAMMD1 (NC_008385), pWDL7::rfp (GQ495894),
Cloning and functional analysis of the dca gene cluster. To amplify
fragments carrying dcaQT and dcaA1A2B, possibly encoding chloroani-
line oxidation, total genomic DNAs from P. putida UWC3(pWDL7::rfp)
and E. coli(pNB8c) were used as the templates in PCRs. The primers
CAGGC-3=) were used to amplify the fragment carrying dcaA1A2B. The
resulting PCR product from pWDL7::rfp was ligated to pBBR1MCS-2
the sequences of the dcaA1A2B genes from pWDL7::rfp and pNB8c were
identical (see Results), a dcaA1A2B clone from pNB8c was not needed.
The HindIII-KpnI fragment containing the dcaA1A2B genes from
pJTP510. The primers cloneAA2-F (5=-GCGGCGAATTCGGTACCGTG
GCAGATGGTTGGGTAATTCG-3=) and cloneAA-R (5=-GGCGCCAAG
used to amplify a fragment carrying dcaQT. After restriction digestion
with EcoRI and HindIII, the resulting product was ligated with pJPC13
(37), forming pJTP800 and pJTP820 for pWDL7::rfp and pNB8c, respec-
tively. All plasmid inserts were verified by DNA sequencing using a Big-
plied Biosystems, Carlsbad, CA). The ability of E. coli DH5? to convert
3-CA to chlorocatechol when containing these cloned dca genes was then
tested using whole-cell biotransformation reactions with mixtures con-
taining 3-CA (0.2 g/liter). The culture supernatant was extracted with
NaOH-washed ethyl acetate, and the extract was analyzed by gas
(31, 39). Products were identified by comparison to chemical stan-
a GFP reporter gene fusion assay the putative promoter region, i.e., the
307-bp and 289-bp tnpA-dcaQ intergenic fragments from plasmid
pWDL7::rfp and pNB8c, respectively, were amplified with the primers
pDcaQF (5=-aaagaattcTTGAACGCTCACTCATGTGC-3=) and pDcaQR
(5=-aaaggtaccGCATAGATGCCGGTTGTTTAG-3=), which have EcoRI
and KpnI sites at their 5= ends (lowercase). Amplicons were cleaved with
EcoRI-KpnI and cloned in front of the promoterless gfp cassette in the
pG-GFP promoter probe reporter plasmid (28) (Table 1). E. coli DH5?
cells with the respective cloned fragments or vector only, were grown
overnight in LB medium supplemented with 100 mg/liter ampicillin
(Amp), transferred into fresh medium and grown to an optical density at
600 nm (OD600) of 0.1 to 0.7. The promoter activity of these cultures was
measured using a Modulus single-tube multimode reader (Promega
Corp., Sunnyvale, CA), and the data are presented as fluorescence units/
OD600unit (FU). Data are mean values from 2 to 4 different clones (3
independent measurements each).
To determine transcriptional activity of the dca genes in C. pinatu-
bonensis JMP228 upon induction with 3-CA, both plasmids pWDL7::rfp
and pNB8c were transferred into C. pinatubonensis JMP228. For total
RNA isolation, cultures grown overnight in LB were diluted 1:20 into
fresh LB medium and grown for 3 h at 30°C with or without 50 mg/liter
3-CA. Culture volumes containing similar cell numbers based on OD600
readings were stabilized with RNAprotect bacterial reagent (Qiagen, Va-
lencia, CA), and RNA was isolated using an RNeasy minikit (Qiagen,
Valencia, CA) according to the manufacturer’s instructions. The purified
RNA was then treated with Turbo DNA-free kit (Applied Biosystems,
Carlsbad, CA) and reverse-transcribed using a high-capacity cDNA re-
verse transcription kit with RNase inhibitor (Applied Biosystems, Carls-
bad, CA) both according to the manufacturers’ instructions. PCR ampli-
fication of a segment of the dcaA1 gene was carried out on cDNA, using
of RNA transcribed, as determined with a Nanodrop ND-1000 spectro-
photometer (Thermo Fisher, Wilmington, DE). The primers used were
NB8c24880 (5=-ATGAATGGGACGACCTGGTG-3=) and NB8c25793r
(5=-AAAGGATCCCAGATTGGGAAAGATGTTGAGG-3=), and the an-
nealing temperature was 65°C. Amplicons were separated on 0.75% aga-
Król et al.
aem.asm.orgApplied and Environmental Microbiology
bands was quantified using Quantity One software (Bio-Rad Laborato-
ries, Hercules, CA).
all plasmid transfer experiments because it is Ilv?and does not grow in
MMO. Plate matings were performed essentially as described previously
(29). Briefly, mixtures of an overnight-grown donor culture of P. putida
UWC3(pWDL7::rfp) and either activated sludge or an overnight grown
recipient culture were placed on LB agar, allowing conjugation to occur
for 12 h. Subsequently, the biomass of these mating mixtures and of neg-
ative controls (separate donor and recipient cultures) was scraped from
the agar surface, and the cells were washed three times by centrifugation
and resuspension in phosphate-buffered saline (PBS) to remove agar and
nutrients. The mating mixtures were used to study the effect of bioaug-
mentation because it was not possible to isolate and stably maintain
transconjugants capable of growing on 3-CA as the sole carbon source.
The washed cell pellets were then resuspended in 1 ml PBS, and added to
125 ml screw-cap Erlenmeyer flasks with 25 ml of MMO, containing ei-
ther 0.16 mM (20 mg/liter) or 0.39 mM (50 mg/liter) of 3-CA as the sole
carbon source. Cell densities were adjusted to equal optical densities, and
samples were removed at 24-h intervals and were prepared for high-
nologies, Santa Clara, CA) with a Phenomenex Prodigy 5 ?m ODS-2
octyldecyl silane column (100 mm by 2 mm) using isocratic 50:50 water-
peaks at 210, 254, and 290 nm, essentially as previously described (16).
rfp and pNB8c have been deposited in the GenBank database under ac-
cession numbers GQ495894 and JF274990, respectively.
General features of plasmids pWDL7::rfp and pNB8c. Determi-
nation of the complete nucleotide sequence of pWDL7::rfp re-
quired a combination of approaches due to its unique and unex-
pected genetic structure. The initially assembled sequence
obtained by shotgun sequencing was not consistent with the plas-
mid restriction fragment profiles and the observed size of uncut
plasmid DNA separated by pulsed-field gel electrophoresis
(PFGE) (data not shown). Results obtained by PCR also con-
firmed the presence of a repeated region (data not shown). By
using these data, the complete genome sequence of plasmid
pWDL7::rfp was reassembled (Fig. 1A). The plasmid is 86,647 bp
long and contains two 22.4-kb inverted repeats, named trans-
poson Tn6063, which are flanked by inverted IS1071 sequences.
red fluorescent protein and kanamycin resistance was found in-
serted into Tn6063 (Fig. 1A). The plasmid genome contains 88
assigned open reading frames (ORFs). The mean G?C content is
63 mol%, but that of the backbone, which consists of plasmid
replication, maintenance/control, and transfer genes, is slightly
(60,421 bp). The automatic sequence assembly in this case was
overall G?C content of 65%, which is very similar to the esti-
by an 18,915-bp segment, which was named Tn6063= because of
its high similarity to Tn6063 on pWDL7::rfp (Fig. 1A). Sixty-four
ORFs were assigned (Fig. 1A).
Plasmids backbone structure and phylogenetic relatedness
sible for conjugative transfer, initiation of vegetative replication,
stable inheritance, and regulatory networks for these functions
brane protein, upf31.7, encoding a site-specific methylase, and
to backbones of the plasmid subgroup IncP-1? (42, 51, 58). The
backbone regions are most similar to those of the IncP-1? plas-
mids pA81 from Achromobacter xylosoxidans A8 (accession no.
AJ515144), and pCNB1 from Comamonas sp. CNB-1 (accession
no. EF079106) (24, 32), both of which also encode proteins in-
volved in catabolic functions (Fig. 2).
Phylogenetic analysis using 24 concatenated protein se-
quences encoded by IncP-1 plasmids confirmed that pWDL7::
rfp and pNB8c belong to the ? subgroup but form a distinct
clade (Fig. 3). Other members of this clade are the catabolic
plasmids pA81 and pCNB1 mentioned above, the cryptic plas-
mid pA1 of Sphingomonas sp. A1 (19), the Acidovorax sp. JS42
plasmid pAOVO02 (GenBank accession no. NC_008766), and
the exogenously isolated multiresistance plasmid pB4 from an
unknown host (50). This new clade has been named IncP-1?-2
by Norberg et al. (36). Very recently, plasmid pAKD26, which
dichlorophenoyacetic acid and catechol degradation (mocp)
genes, was also shown to be a member (not shown in Fig. 3)
(43). Thus, plasmids pWDL7::rfp and pNB8c have a backbone
that is divergent from that of the archetypal IncP-1? plasmid
R751 but very similar to that of other catabolic plasmids in the
orientation of the two backbone regions. Because the two Tn6063
transposons are identical, it was not possible to establish the final
orientation of the backbone regions simply based on the assem-
bled DNA sequence. Therefore, pWDL7::rfp DNA was digested
with the restriction enzymes PsiI and SnaBI, and the resulting
fragments were separated by PFGE. In the pWDL7::rfp backbone
sequence, a single SnaBI site is located between upf30.5 and
and precedes relE (stbE) (Fig. 4B). The resulting restriction frag-
ments in this case should be 39,189 and 47,458 bp long (form A).
If the backbone regions were inverted (form B), different DNA
fragments should be observed (20,932 and 65,715 bp). Surpris-
ingly, PFGE yielded four DNA fragments in identical molar ratio,
E. coli strain from which the plasmid DNA was purified before
rfp. Thus, the sequence assembled and deposited in the GenBank
database represents only one of these two orientations (form A).
Accessory genetic elements. Most IncP-1 plasmids described
to date carry catabolic genes, mercury resistance determinants, or
multidrug resistance determinants, which are usually located be-
and carries putative 2,4-
Plasmid-Mediated Chloroaniline Degradation
February 2012 Volume 78 Number 3aem.asm.org 831
to be occupied by identical copies of transposon Tn6063, inserted
in opposite orientations (Fig. 1A). The two Tn6063 insertion sites
did not share any sequence similarity, but the copy located at site
2 is flanked by a pair of 5-bp direct repeats in form A of the
transposon Tn6063= is inserted in site 1. It is almost identical to
Tn6063 on pWDL7::rfp except for slight differences in the dca
operon sequence (see below). This suggests that the first transpo-
sition of Tn6063 into the pWDL7::rfp plasmid occurred at site 1,
followed by recent insertion into site 2 (Fig. 4A).
Characterization of the Tn6063 transposon. Tn6063 has a
complex structure composed of two insertion sequences (IS1071
and IS1071a) flanking a region that contains two other insertion
sequences, IS21 and IS66, as well as an operon of catabolic genes,
named dca by homology to recently identified dca genes from
Variovorax sp. (5, 8). Moreover, the mini-Tn5 element that was
used to mark pWDL7::rfp was found within the 3= end of the
second IS1071-like transposase gene. The transposases of the
flanking IS1071 and IS1071a elements share 78% identity (88%
similarity) without gaps at the protein level. The IS1071 element
has two 108-bp long inverted repeat sequences (IR3L and IR3R),
while IS1071a seems to contain only the left-side long inverted
repeat (IRL; 110 bp) but not the corresponding right inverted
to each other (Fig. 1B).
The operon that is likely involved in the degradation of chlo-
roaniline (dca) by encoding a peripheral (upper) degradation
pathway, contains six genes. Due to the extremely high DNA se-
quence similarity with the recently annotated dca genes of the
linuron degrader Variovorax sp. SRS16, shown to be induced by
FIG 1 Genetic map of the IncP-1? plasmids pWDL7::rfp (outer circle) and pNB8c (inner circle) (A) and comparison of IS1071 and IS1071a inverted repeats
located on Tn6063 (B). (A) Coding regions are shown by arrows indicating the direction of transcription. The regions encoding Tra1 (tra) and Tra2 (trb), the
differentiated by color. The repeated 22.3-kb accessory gene region downstream of either the conjugative transfer gene traC or the replication initiation protein
trfA in pWDL7::rfp consists of (i) the catabolic dca genes (degradation of chloroaniline), (ii) mini-Tn5 (KmrCmrrfp), and (iii) insertion sequences IS1071,
IS1071a, IS66, and IS21 and other transposases. (B) Differences in IR sequences are marked in black and blue.
Król et al.
aem.asm.org Applied and Environmental Microbiology
3,4-dichloroaniline (3,4-DCA) (5), we also named the operon on
pWDL7:rfp and NB8c dca. It was previously shown that aniline
dioxygenases consist of multiple components: proteins homolo-
gous to glutamine synthetase, glutamine amidotransferase, large
and small subunits of the terminal dioxygenase, and a reductase
these systems were first described, these components are encoded
by, respectively, tdnQ-tadQ, tdnT-tadT, tdnA1-tadA1, tdnA2-
tadA2, and tdnB-tadB (18, 30). The genes dcaQ, -T, -A1, -A2, -B,
and -R on pWDL7::rfp and pNB8c show high sequence similarity
thought to code for conversion of (chloro)aniline to (chloro)cat-
echols (5, 17, 18, 35, 59). No homologs of aromatic ring cleavage
pathway genes were identified on either pWDL7::rfp or pNB8c.
Functional analysis of dca genes encoded by pNB8c and
differences in catabolic activity had been previously observed.
While pWDL7 was shown to confer 3-CA and 3,4-DCA transfor-
mation after conjugative transfer in C. pinatubonensis JMP228n
(14), pNB8c conferred only the ability to transform nonchlori-
nated aniline in an isogenic strain, even though its original host,
NB8c, degraded 3-CA (7). To determine the molecular basis of
these differences in catabolic activity between the two plasmids,
the dca genes and their promoter regions, as well as their func-
tions, were compared in detail. Discrete differences were revealed
in two areas of the dca operons: the dcaQT genes and the dca
promoter region (Fig. 5A and B).
First, on pNB8c, three single-nucleotide polymorphisms
(SNPs) in the dcaQ gene caused amino acid substitutions, and a
start codon 75 bp downstream compared to the start site in
pWDL7::rfp. These changes may affect the activity of the DcaQ
tional and sufficient to allow transformation of 3-CA into
4-chlorocatechol (4-CC) and (ii) that the effect of the SNPs in
dcaQT did not affect that function. The dcaQT genes from both
plasmids and the dcaA1A2B from pWDL7::rfp were cloned sepa-
activity was measured in E. coli (Fig. 5B; Table 1). Since the
dcaA1A2B sequences from both plasmids are identical, the dca
genes on pJTP510 from pWDL7::rfp are representative of both.
The E. coli DH5? strains carrying either plasmids pJTP800 and
pJTP510 (expressing dcaQT and dcaA1A2B from pWDL7::rfp) or
plasmids pJTP820 and pJTP510 (expressing dcaQT from pNB8c
and dcaA1A2B from pWDL7::rfp/pNB8c) were both able to con-
vert 3-CA to 4-CC, as judged by GC-MS analysis, with no observ-
able differences. In contrast, E. coli carrying only the dcaQT genes
3-CA. However, the cloned dcaA1A2B genes alone [in E. coli
DH5?(pJTP510)] conferred chloroaniline dioxygenase activity,
as judged by the formation of 4-CC (Fig. 5B). These results indi-
cate that in E. coli, the dcaA1A2B genes are sufficient for conver-
coding regions of the two plasmids do not explain the drastic
differences in the ability of pWDL7::rfp and pNB8c to confer this
FIG 2 Comparison of pWDL7::rfp with Achromobacter xylosoxidans A8 plasmid pA81 and plasmid pCNB1 from Comamonas sp. CNB-1 (the sequence was
reoriented to start from the trfA stop codon). The shaded connecting bands show direct and inverted homologies with different percent similarities. Core
functional regions and accessory genes are differentiated by patterns (because of the high sequence similarity, plasmid pNB8c is not shown).
FIG 3 Phylogenetic tree of IncP-1 plasmids showing the relationship of
shared backbone genes from 26 plasmids were aligned individually and then
concatenated into a single alignment. RAxML-VI-HPC was used to infer a
maximum likelihood phylogeny for the aligned sequences. The phylogeny
with the largest likelihood was chosen from 100 iterations that altered the
input order of each concatenated sequence.
Plasmid-Mediated Chloroaniline Degradation
February 2012 Volume 78 Number 3aem.asm.org 833
FIG 4 Proposed mechanism of transposition leading to duplication of the accessory regions (A) and genetic maps of the two forms of the pWDL7::rfp plasmid
(B). (B) Coding regions are shown by arrows indicating the direction of transcription. The Tra1 and Tra2 regions (tra and trb), the replication (rep) module
(trfA-ssb), the central control region (ctl), encoding regulatory and stability functions (from relE to kfrC), and the partition gene parC, as well as the two copies
of the Tn6063 transposon, are differentiated by gray scale. SnaBI/PsiI restriction fragments are marked A1, A2 (form A), B1, B2 (form B). (C) PFGE gel of
pWDL7::rfp DNA cut with SnaBI (lane 2) and SnaBI/PsiI (lane 3); lanes 1 and 4 contain low-range PFGE marker (New England BioLabs, Ipswich, MA) and
high-molecular-weight DNA marker (Invitrogen Life Technologies, Carlsbad, CA), respectively.
Król et al.
aem.asm.orgApplied and Environmental Microbiology
Second, compared to the sequence of pWDL7::rfp, deletions
dcaQ start codon (Fig. 5A). In silico analysis revealed the presence
of a hypothetical E. coli ?70-like promoter, and the mutations in
effect of these deletions, we used two approaches. In a first ap-
cloned in front of a promoterless gfp gene (Table 1). While the
pWDL7::rfp fragment showed strong promoter activity (dcaQ,
30,557 ? 13,276 FU), the activity of the pNB8c fragment was as
low as that of the control E. coli strain with the insert-free vector
(?700 FU) (Fig. 5A). As these results were obtained in an E. coli
JMP228, in which catabolic activity differences were previously
observed. Therefore, we compared the transcriptional activity of
the dca operons from both plasmids in that host in the presence
and absence of 3-CA. Repeated assays consistently showed that
of 3-CA. Yet when the cultures were grown with 3-CA for several
consistent increase in transcript levels compared to uninduced
cultures, but JMP228(pNB8c) did not (data not shown). These
results suggest that the modified segment of the pNB8c pdca pro-
moter region was insufficient for regulation of the dca operon by
3-CA and thus likely explains the previously shown inability of
pNB8c to confer 3-CA conversion in JMP228gfp (7).
Potential of pWDL7::rfp in bioaugmentation of activated
sludge. Since plasmid pWDL7::rfp carries two copies of a fully
functional dca gene cluster, it could be useful in bioaugmentation
projects to accelerate removal of 3-CA from polluted waters or
soils by transferring the genetic information to indigenous bacte-
ria (56). The apparent lack of expression of the dca genes on
plasmid less useful for such applications, and it was therefore not
analyzed further. We tested the ability of pWDL7::rfp to transfer
3-CA degradation capacity from P. putida UWC3 to a mixed bac-
terial community from the activated sludge of a wastewater treat-
ment plant in plate matings. In parallel to the activated sludge
communities, pure cultures of Delftia acidovorans ATCC 15668
and Comamonas testosteroni ATCC 11996, which are unable to
degrade 3-CA, were used as control recipients for the plasmid,
because other strains of these species have been reported to be
involved in 3-CA degradation (6, 7, 9, 13, 22, 49, 63).
Mating mixtures were used to study the effect of bioaugmen-
tation because it was not possible to isolate and stably maintain
transconjugants capable of growing on 3-CA as the sole carbon
source. When activated sludge samples bioaugmented with P.
putida UWC3(pWDL7::rfp) were suspended in MMO with 0.16
source, little degradation occurred over the first 5 days. However,
source on day 7, 3-CA was rapidly removed, with no 3-CA re-
maining after 9 days (Fig. 6). Bioaugmented cultures of D. acido-
vorans ATCC 15668 removed 100% of 3-CA after 9 days (Fig. 6).
In contrast, mixtures with C. testosteroni ATCC 11996 as the re-
uninoculated controls showed little 3-CA degradation until day
11, after which the 3-CA concentration rapidly decreased in all
ing with 3-CA as a sole carbon and energy source in mineral salts
medium. Therefore, accelerated 3-CA degradation activity in
acquired and expressed the plasmid-encoded degradation genes.
the possible accumulation of degradation products. Overall, the
(black arrow). Gray boxes represent computer predicted ?35 and ?10 boxes of a hypothetical E. coli ?70-like promoter. (B) Organization of the dca genes and
3-CA degradation activity of derived clones (gray dcaQT arrows indicate pNB8c genes). Bars represent cloned regions of the dca operon. The chloroaniline
dioxygenase activity of E. coli DH5? cells carrying the indicated plasmids, as determined by the formation of 4-CC, is shown.
Plasmid-Mediated Chloroaniline Degradation
February 2012 Volume 78 Number 3aem.asm.org 835
addition of a donor carrying pWDL7::rfp to activated sludge de-
creased the lag time for 3-CA removal by 5 to 7 days. Such a time
“saving” could be critical in bioaugmentation applications. Thus,
providing the genes encoding the upper pathway for 3-CA degra-
dation to a wastewater treatment reactor may greatly accelerate
partial or complete chloroaniline removal, depending on the
composition of the activated sludge community and the presence
of a readily available carbon source.
We present the complete genome sequences of two plasmids,
the IncP-1? plasmid group and have a broad host range, as they
are able to transfer between beta- and gammaproteobacteria.
can explain their different abilities to confer chloroaniline degra-
the catabolic gene products of both plasmids were able to convert
3-CA to 4-CC but that only the dca genes on pWDL7::rfp and not
those on pNB8c were induced by 3-CA, likely due to changes in
the pNB8c dca promoter region. Plasmid pWDL7::rfp was also
shown to have potential as a bioaugmentation agent to augment
microbial communities of wastewater treatment plants with the
catabolic functions needed to convert chloroanilines.
copies of Tn6063, one in each of the two expected accessory gene
caused by intramolecular replicative transposition (Fig. 4). This
mechanism can create two inversely oriented copies of a replica-
tive transposon in one circular DNA molecule. It is accompanied
transposon and the site where the transposon was originally lo-
cated (10). In plasmid pWDL7, such an event could have been
mediated by the IS1071-type transposase acting on the inverted
repeats IRL and IR3L. Therefore, it is plausible that plasmid form
form A was subsequently generated by homologous recombina-
tion between the duplicated transposon regions. Sota et al. (45)
duplication. The presence of 5-bp direct repeats flanking one of
the Tn6063 copies strongly suggests the occurrence of replicative
transposition. Based on the characteristics of IS1071, which can
actively transpose only in specific bacterial species, it is highly
unlikely that the Tn6063 duplication occurred in E. coli (45). To
test this hypothesis, we confirmed the presence of two copies of
amplicons were identical in the cases of the original and marked
finished C. testosteroni WDL7 genome sequencing project also
and D. Springael, personal communication). The presence of two
identical catabolic transposons on one plasmid seems rare but
illustrates the potential involvement of transposable elements in
We showed that the dcaA1A2B genes alone are sufficient for
chloroaniline dioxygenase activity. These results are different
from those of studies of the aniline dioxygenase from P. putida
UCC22(pTDN1) and Frateuria sp. ANA18, which showed that
strains of E. coli required all of the cloned tdnQ, -T, -A1, -A2, and
used a very sensitive assay for the detection of 4-CC and do not
know if, in the natural host, sufficient conversion of 3-CA would
also necessary to determine if the presence of a second copy of
Tn6063 on pWDL7::rfp significantly increases the enzyme con-
centration in the cell and therefore the rate of conversion of 3-CA
to 4-CC, compared to a plasmid with a single Tn6063 copy like
pNB8c compared to pWDL7::rfp was associated with lack of in-
duction of dca transcription in the host strain C. pinatubonensis
JMP228 compared to the strain carrying pWDL7::rfp. These data
pinatubonensis JMP228gfp, while its native host was able to de-
grade 3-CA (7). The mechanism by which the native host of
pNB8c effectively metabolized 3-CA is not known. This finding
shows how subtle genetic differences in catabolic operons can
drastically affect the degradation phenotypes conferred by two
very similar plasmids.
Previous results suggested that the native plasmid pWDL7
conferred complete mineralization of 3-CA to strain JMP228n
(14), yet the marked derivative pWDL7::rfp does not contain any
chlorocatechol degradation genes. Due to size differences of plas-
mid pWDL7 in different archived clones of JP228n(pWDL7), it is
currently unclear whether that plasmid contained the complete
degradation pathway before it was marked and transferred to
determination of the original pWDL7 host, C. testosteroni WDL7
(Albers and Springael, personal communication), will be able to
address this question.
Our results suggest that plasmid pWDL7::rfp might success-
fully bioaugment wastewater treatment plants by accelerating the
removal of recalcitrant chloroanilines. Even though 3-CA was
eventually removed by the activated sludge itself, the acceleration
FIG6 Degradation of 0.16 mM 3-CA by pure and mixed cultures of P. putida
UWC3(pWDL7::rfp) (?), activated sludge (Œ), D. acidovorans ATCC 15668
(’), P. putida UWC3(pWDL7::rfp) mated with activated sludge (?), and P.
putida UWC3(pWDL7::rfp) mated with D. acidovorans ATCC 15668 (?) and
experiment was repeated three times with comparable results.
Król et al.
aem.asm.orgApplied and Environmental Microbiology
of degradation by almost a week can be of great benefit in a real
treatment reactor. As more water can be treated in a given period
of time, the plant footprint can be reduced, which translates into
great cost savings. This accelerated removal of a chlorinated aro-
matic compounds is in line with previous reports of successful
bioaugmentation of soils and wastewater treatment systems by
conjugal transfer of plasmids that enable degradation of chlori-
mentation by conjugative transfer of the IncP-1 plasmid pNB2 to
activated sludge bacteria might enhance chloroaniline transfor-
mation (4). Like pWDL7::rfp, this plasmid is thought to contain
7). Therefore, both in that study and ours, the genomes of strains
that can use 3-CA as the sole carbon source upon acquisition of
age pathway to allow chlorocatechol metabolism. The efficient
removal of 3-CA by the mating mixture of UWC3(pWDL7::rfp)
D. acidovorans strains have been shown to possess the necessary
lower-pathway genes needed for mineralization of 3-CA (22).
In summary, based on their complete genome sequences, the
respectively, two copies and one copy of a chloroaniline dioxyge-
nase gene cluster that encodes only the upper pathway of chloro-
aniline degradation. Moreover, comparison of the two plasmids’
gests that promoter differences can explain the lack of catabolic
activity conferred by pNB8c outside its native host. Finally, bio-
resulted in accelerated 3-CA removal, suggesting the plasmid’s
potential in aiding 3-CA cleanup by wastewater treatment plants.
This work was supported by grants EF-0627988 from the NSF Microbial
Genome Sequencing Program, and NIH grants R01 GM073821 from the
National Institute for General Medical Sciences, and P20RR16448 and
P20RR016454 (COBRE and INBRE programs) from the National Center
for Research Resources. Support was also provided by the University of
California Toxic Substances Research and Teaching Program and Eco-
toxicology Lead Campus Program to S.W. and by NSF grant MCB-
1022362 to R.E.P.
We are grateful to the U.S. Department of Energy Joint Genome In-
stitute for providing the draft plasmid genome sequences (with special
thanks to Brian Foster, Alla Lapidus and Kerry Barrie). Their work is
supported by the Office of Science of the U.S. Department of Energy
assistance with HPLC method development, G. Van der Auwera for gen-
erating Fig. 2 and for useful discussions and suggestions, N. Boon for
providing plasmid pNB8c, and JCVI for offering the JCVI Annotation
Service, which provided us with automatic annotation data and the man-
ual annotation tool Manatee.
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