The whole genome sequence of Sphingobium chlorophenolicum L-1: insights into the evolution of the pentachlorophenol degradation pathway.

Abstract

Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the pathway were acquired from the most recent common ancestor of these two bacteria.

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The Whole Genome Sequence of Sphingobium
chlorophenolicum L-1: Insights into the Evolution of the
Pentachlorophenol Degradation Pathway
Shelley D. Copley1,*, Joseph Rokicki1, Pernilla Turner2, Hajnalka Daligault3, Matt Nolan4, and Miriam Land5
1Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder
2The Cooperative Institute for Research in Environmental Sciences, University of Colorado at Boulder GeoSynFuels LLC, Golden,
Colorado
3Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico
4Joint Genome Institute, Walnut Creek, California
5Oak Ridge National Laboratory, Oak Ridge, Tennessee
*Corresponding author: E-mail: shelley.copley@colorado.edu.
Accepted: 8 December 2011
Data depostion: GenBank accession numbers for the replicons of S. chlorophenolicum are given in table 1.
Abstract
Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced
the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the
genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental
adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have
compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that
degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway
were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the
pathway were acquired from the most recent common ancestor of these two bacteria.
Key words: horizontal gene transfer, biodegradation, enzyme evolution, pentachlorophenol hydroxylase, tetrachlorohy-
droquinone dehalogenase, tetrachlorobenzoquinone reductase.
Introduction
In the early 20th century, consumers embraced the concept
of ‘‘better living through chemistry.’’ Massive quantities of
chlorinated anthropogenic chemicals such as dichlorodiphe-
nyltrichloroethane(DDT),lindane,pentachlorophenol(PCP),
atrazine, tetrachloroethylene, and polychlorinated biphen-
yls (PCBs) were introduced into the environment with little
concern over their ultimate fate. Many of these compounds
persist in the environment and have adverse effects on eco-
systems. Use of some of these compounds (e.g., DDT and
PCP) is now restricted or prohibited in the United States,
although some are still used in developing countries.
The introduction of anthropogenic chemicals into the
environment imposes selective pressures that can foster
evolution of novel pathways that allow microbes to access
new sources of carbon, nitrogen, or phosphorus and/or to
detoxify toxic compounds. However, degradation of anthro-
pogenic chemicals is often inefficient because microbes have
not yet evolved enzymes that efficiently catalyze the steps
needed to convert anthropogenic compounds into metabo-
lites in central carbon metabolism (Copley 2009).
The pesticide PCP is listed as a Priority Pollutant by the US
EPA due to its toxicity and persistence in the environment.
AnumberofmicrobescapableofmineralizingPCPhavebeen
isolated from contaminated sites (Saber and Crawford 1985;
Apajalahti and Salkinoja-Salonen 1987; Schenk et al. 1990;
Uotilaetal.1991;Radehaus1992;Karnetal.2010).Thebest
studied of these is Sphingobium chlorophenolicum (Saber
and Crawford 1985; Steiert and Crawford 1986; Crawford
ª The Author(s) 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/
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and Ederer 1999; Takeuchi et al. 2001). Sphingobium chlor-
ophenolicum has patched together a poorly functioning
pathway (see fig. 1) that allows mineralization of PCP,
although degradation is slow and the bacterium is unable
to grow at high concentrations of PCP (Dai and Copley
2004). The first three enzymes in the pathway are unusually
ineffective. PCP hydroxylase (PcpB) has a very low kcatof
0.02 s?1and shows substantial uncoupling; the enzyme fre-
quently fails tohydroxylate the substrate and instead releases
H2O2as the C4a-hydroperoxyflavin intermediate decomposes
(Hlouchova K, Rudolph J, Pietari JMH, Behlen LS, Copley SD,
unpublished data). Tetrachlorobenzoquinone (TCBQ) reduc-
tase (PcpD) is also rather inefficient, with a turnover rate of
only 0.7 s?1at 50 lM TCBQ (Dai et al. 2003). Tetrachlorohy-
droquinone (TCHQ) dehalogenase (PcpC) is subject to pro-
found substrate inhibition; binding of its aromatic substrates
to the active site before completion of the catalytic cycle inter-
fereswiththefinalsteprequiredtoregeneratethefreeenzyme
(WarnerandCopley2007a).Thesefindingsareconsistentwith
the hypothesis that the enzymes in the PCP degradation path-
way have not yet evolved to the level of function typical of
most metabolic enzymes. Thus, S. chlorophenolicum provides
a window on the process of assembly of a new pathway at
a relatively early stage in its evolution.
We recently sequenced the genome of S. chloropheno-
licum L-1. Analysis of this sequence and comparison with
thesequenceofthecloselyrelatedSphingobiumjaponicum,
which degrades lindane (Nagata et al. 2007), provides
insights into the origins of the PCP degradation enzymes.
The rDNA genes of S. chlorophenolicum and S. japonicum
share 97% identity. The phylogenetic distance between
S. chlorophenolicum and S. japonicum is ideal to facilitate
identification of genes that were present in the most recent
commonancestorofthese twoSphingomonads,asproteins
involvedincoreprocessesshow.80%pairwiseidentitiesat
the amino acid level.
Our analysis suggests that the first three enzymes in the
pathway were acquired by S. chlorophenolicum by horizon-
tal gene transfer (HGT) after it diverged from S. japonicum.
In contrast, the last two enzymes in the pathway were pres-
ent in the most recent common ancestor of S. chloropheno-
licum and S. japonicum. None of the genes encoding the
PCP degradation enzymes arose by recent duplication
and divergence of genes within S. chlorophenolicum. The
genes occur in two disparate parts of the genome and have
not yet been integrated into a compact and consistently
regulated operon.
Materials and Methods
Isolation of Genomic DNA from S. chlorophenolicum L-1
A sample of S. chlorophenolicum L-1 was originally depos-
ited tothe American typeCulture Collection anddesignated
ATCC 39723. The initial characterization of the enzymes in
the PCP degradation pathway was done with the ATCC
39723 strain (Orser and Lange 1994; Copley 2000; Dai
et al. 2003; Warner and Copley 2007b). However, the ATCC
39723 strain lost the ability to degrade PCP, so a second
sample of S. chlorophenolicum L-1 was deposited and
designated ATCC 53874. Genomic DNA was isolated from
this strain.
A 5 ml culture of ATCC medium 1687 (Flavobacterium
medium) containing 5 lM PCP was inoculated with a colony
from a fresh agar plate of half-strength tryptone soy broth
PCP
OH
Cl
Cl
Cl
Cl
Cl
OH
Cl
Cl
OH
Cl
Cl
OH
Cl
Cl
OH
H
Cl
OH
Cl
H
OH
H
Cl
OH
H
Cl
H
Cl
O
O
OH
O
Cl
Cl
O
Cl
Cl
OH
H
Cl
H
O
O
O
OH
OH
H
H
O
O
O
OH
PcpB
PcpD
PcpC
PcpC
PcpAPcpE
HGT event
HGT event
recruitment of pre-existing enzymes
OH
H
H
O
O
O
OH
PcpE
spont.
FIG. 1.—PCP degradation pathway. PcpB, pentachlorophenol hydroxylase; PcpD, tetrachlorobenzoquinone reductase; PcpC, tetrachlorohydro-
quinone dehalogenase; PcpA, 2,6-dichlorohydroquinone dehalogenase; PcpE, maleylacetate reductase; HGT, horizontal gene transfer.
Evolution of the Pentachlorophenol Degradation Pathway
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containing 50 lM PCP and incubated with shaking at 30 ?C
overnight. One milliliter aliquots of cells were harvested
by centrifugation at 16,100 ? g for 1 min. Genomic DNA
was isolated using the Aquapure Genomic DNA kit (Bio-
Rad, Hercules, CA). Cell pellets from 1 ml of culture were
resuspended in 300 ll Genomic DNA Lysis Solution. The so-
lution was incubated at 80 ?C for 5 min to lyse the cells.
A solution ofRNase A (1.5 ll, 4 mg/ml) was added,and tube
wasinverted25timesbeforeincubationat37?Cfor45min.
The temperature of the sample was adjusted to room tem-
perature and 100 ll of Protein Precipitation Solution was
added.Thesamplewasvortexedfor20sandthensubjected
to centrifugation at 13,000 ? g for 3 min to remove cellular
debris. The supernatant containing DNA was transferred to
a clean 1.5 ml tube, incubated on ice for 5 min, and then
subjected to centrifugation again. The supernatant was
then transferred to a clean 1.5-ml tube containing 300 ll
100% isopropanol to precipitate the DNA. The tube was
inverted 50 times. The DNA was pelleted by centrifugation
at 13,000 ? g for 1 min. The supernatant was poured off,
and the pellet was washed with 300 ll 70% ethanol. The
ethanol was poured off and residual ethanol removed by
draining the tube onto filter paper for 10–15 min. The pellet
was dissolved in 100 ll 10 mM Tris–HCl, pH 7.5, at 65 ?C for
5 min and then at room temperature overnight. The quality
of the DNA was checked on a 0.8% agarose 1X TAE
gel alongside a 1 kb DNA Extension Ladder (Invitrogen,
Carlsbad, CA), and the concentration was measured on
a NanoDrop spectrophotometer.
Genome Sequencing
The draft genome of S. chlorophenolicum L-1 was gener-
ated at the DOE Joint genome Institute (JGI) using a combi-
nation of Illumina (Bennett 2004) and 454 technologies
(Margulies et al. 2005). Three libraries were constructed
and sequenced: an Illumina GAii shotgun library that gen-
erated 40,283,193 reads totaling 3,061 Mb; a 454 Titanium
standard library that generated 302,660 reads and a paired
end 454 library with an average insert size of 11.26 ± 2.81
kb that generated 174,361 reads. In total, 176.8 Mb of 454
data were obtained. General aspects of library construction
and sequencing performed at the JGI can be found at http://
www.jgi.doe.gov/. The initial draft assembly contained 79
contigs in one scaffold. The 454 Titanium standard data
and the 454 paired end data were assembled with Newbler,
version 2.3. The Newbler consensus sequences were com-
putationally shredded into 2 kb overlapping fake reads
(shreds). Illumina sequencing data was assembled with VEL-
VET, version 0.7.63 (Zerbino 2008), and the consensus
sequences were computationally shredded into 1.5 kb
shreds. The 454 Newbler consensus shreds, the Illumina
VELVET consensus shreds, and the read pairs in the 454
paired end library were integrated using parallel phrap,
version SPS—4.24 (High Performance Software, LLC). The
software Consed (Ewing and Green 1998; Ewing et al.
1998; Gordon et al. 1998) was used in the finishing process.
Illumina data were used to correct potential base errors and
increaseconsensusqualityusingthesoftwarePolisherdevel-
oped at JGI (Lapidus A, unpublished data). Possible misas-
semblies were corrected using gapResolution (Han C,
unpublished data) or Dupfinisher (Han 2006) or by sequenc-
ing cloned bridging PCR fragments. Gaps between contigs
were closed by editing in Consed, by PCR, and by Bubble
PCR (Cheng J-F, unpublished data) primer walks. A total
of 87 additional finishing reactions (either sequencing of
bridging PCR fragments or primer walking) were necessary
to close gaps and to raise the quality of the finished
sequence. The total size of the genome is 4,573,221 bp.
The final assembly is based on 176.8 Mb of 454 draft data
that provides an average 36? coverage of the genome and
3,553 Mb of Illumina draft data that provides an average
790? coverage of the genome.
GeneswereidentifiedusingProdigal(Hyattetal.2010)as
part of the Oak Ridge National Laboratory genome annota-
tion pipeline, followed by a round of manual curation using
the JGI GenePRIMP pipeline (Pati et al. 2010). The predicted
coding sequences were translated and used to search the
National Center for Biotechnology Information nonredun-
dant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG,
COG,andInterProdatabases.Thesedatasourceswerecom-
bined to assert a product description for each predicted pro-
tein. Noncoding genes and miscellaneous features were
predictedusingtRNAscan-SE(LoweandEddy1997),RNAm-
mer (Lagesen et al. 2007), Rfam (Griffiths-Jones et al. 2003),
TMHMM (Krogh et al. 2001), and signalP (Bendtsen et al.
2004).
Results and Discussion
The S. chlorophenolicum Genome
The S. chlorophenolicum genome consists of two chromo-
somes and a plasmid (see fig. 2). A summary of the charac-
teristics of each replicon is provided in table 1. Like many
bacteria such as Burkholderia pseudomallei (Holden et al.
2004) and Vibrio cholerae (Heidelberg et al. 2000) that con-
tain multiple replicons, S. chlorophenolicum has a dominant
chromosome that carries most of the genes for core pro-
cesses. We generated a list of genes involved in replication,
transcription, translation, cell division, peptidoglycan bio-
synthesis, and core metabolic processes (including glycoly-
sis, the pentose phosphate pathway, the TCA cycle, electron
transport, and biosynthesis of amino acids, nucleotides, and
cofactors) (see supplementary table 1, Supplementary Ma-
terial Online). Figure 3 shows the distribution of genes be-
tween the two chromosomes. If genes encoding core
functions were randomly distributed between the chromo-
somes,wewouldexpect354(72%)tobeonchromosome1
Copley et al.
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and 138 (28%) to be on chromosome 2. In fact, 442 core
genes (90%) are on chromosome 1 and only 50 (10%) are
on chromosome 2. This deviation from the expected distri-
bution is highly significant by chi-square analysis (P value 5
2.2 ? 10-16). An odd collection of essential enzymes, includ-
ingafewgenesforcentralcarbonmetabolism,twosubunits
ofRNApolymerase,somecomponentsoftheelectrontrans-
fer chain, and a few genes for cofactor biosynthesis are
found on chromosome 2 rather than on chromosome 1.
However, most of the genes on chromosome 2 appear to
be involved in environmental adaptation. Chromosome 2
carries a number of genes predicted to encode transporters
for sugars and metal ions and enzymes involved in degrada-
tion of various sugars, short-chain fatty acids, and aromatic
compounds. All the PCP degradation genes are found
on chromosome 2. A secondary chromosome with a low
density of essential genes may serve as a convenient stor-
age depot for genes acquired by HGT, as integration of
newly acquiredDNA is not likely to disrupt an essential func-
tion. Chromosome 2 also contains two genes encoding
proteins related to ParB, which is involved in plasmid parti-
tioning. These features of chromosome 2 are consistent
with the proposal that bacterial secondary chromosomes
have arisen by intragenomic transfer of essential genes,
including rRNA genes, to a plasmid (Slater et al. 2009).
HGT is rampant among microbes and is known to play
a major role in acquisition of resistance to or degradation
of toxic compounds, including antibiotics. We examined
the S. chlorophenolicum genome for features indicative
of mobile genetic elements. As mentioned above, S. chlor-
ophenolicum contains one plasmid. The genome appears to
contain one integrated prophage; a clusterof phage-related
FIG. 2.—Circle diagram of the replicons of Sphingobium chlorophenolicum. The second circle in each replicon indicates the locations of PCP
degradation genes in black, phage genes in blue, and transposon-related genes in red. The third circle shows locations of genes that have a close
homolog in Sphingobium japonicum (.80% identity over .90% of the S. chlorophenolicum sequence). The fourth circle shows GC content. Red
indicates sequences with GC content ,64% and blue indicates sequences with GC content .64%. Light gray lines are placed at intervals of one
standard deviation from the mean of 63.8%. (One standard deviation 5 0.027%; GC content was calculated by averaging over a 10-kb window and
sliding that window in 1-kb increments.) Diagram was generated using Circos (Krzywinski et al. 2009).
Table 1
Genome Statistics
Length (bp) GC ContentORFs rRNA Operons
Chromosome 1 (NC_015593)
Chromosome 2 (NC_015594)
Plasmid (pSPHCH01) (NC_015595)
3080818
1368670
123733
0.64
0.64
0.65
2,940
1,159
125
1
2
0
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genes (including a major capsid protein, major tail protein,
phage portal protein, pro-head peptidase, and some con-
servedphageproteinsofunknownfunction)isfoundonchro-
mosome 1 (genes Sc_00004260-00004380).Curiously, seven
isolated genes annotated as ‘‘phage integrase family’’
genes are found on both chromosomes 1 and 2 (Chr1:
Sc_00026840, Sc_00029370, Sc_00022480, Sc_00028520,
Sc_00012030; Chr 2: Sc_00031410, Sc_00030440). These
genes have anomalously low GC content (0.48–0.58)
and are not closely related to each other. Only one
(Sc_00026840) is found in the vicinity of a prophage gene
(a CP4-57 regulatory protein). Two (Sc_00017260 and
Sc_0003440)areadjacenttotransposasegenes.Thegenome
also carries 27 sequences annotated as ‘‘transposase,’’
‘‘transposase/integrase core domain,’’ or ‘‘transposase and
inactivated derivatives’’ (16 on chromosome 1, 9 on chro-
mosome2,and2ontheplasmid).Theseproteinsare related
to transposases found in several different families of inser-
tion elements and in Tn3family transposons. Thus, like most
microbialgenomes,thegenomeofS.chlorophenolicumdis-
plays evidence of continual onslaught by mobile genetic
elements. However, the PCP degradation genes show no
association with any of these elements.
New genes that can help microbes survive and grow in
the face of selective pressure from environmental toxins
can also arise by gene duplication and divergence (Hughes
1994; Bergthorsson et al. 2007). We carried out a Blast
search of the S. chlorophenolicum genome against itself
to identify genes that are nearly identical and may have
arisen by recent gene duplication. Only 38 proteins have
.90%sequenceidentitytoanotherproteininS.chlorophe-
nolicum. Of these, 13 are related to transposases; three
of these genes are found in three identical copies scattered
throughout the genome. Although the presence of highly
similar transposase genes is not unexpected, there is little
rhyme or reason to the identities and locations of the
remaining duplicated genes. Seven genes annotated as
encoding 2-hydroxychromene-2-carboxylate
a short-chain alcohol dehydrogenase, a hypothetical pro-
tein, a nucleoside-diphosphate-sugar epimerase, an arabi-
nose efflux permease, a Zn-dependent dipeptidase, and
an outer membrane receptor protein are present between
positions 667462 and 675176 on the—strand of chromo-
some 2. A second copy of five of these genes are found
in a cluster on the þ strand of chromosome 2 between
positions 1036969 and 1030812. Copies of the first two
genes in the cluster are found together in a different loca-
tion on chromosome 2. Nearly identical copies of the genes
encoding the alpha and beta subunits of the E1 component
of pyruvate dehydrogenase are found on chromosomes 1
and 2 and nearly identical copies of a gene annotated as
anacyl-CoA synthetase/AMP-acidligase II are foundin chro-
mosome 2 but surrounded by completely different genes.
The utility of these duplicated genes and the processes lead-
ing to their location in distant parts of the genome are not
clear. One possibility is that they were not actually dupli-
catedinS.chlorophenolicumbutratheracquiredbyintegra-
tion intothegenome ofDNA fragmentstaken upfromlysed
cells of S. chlorophenolicum or closely related Sphingomo-
nads. Notably, none of the PCP degradation genes is found
among the set of highly similar genes.
isomerase,
Comparison of the Genomes of S. chlorophenolicum
and S. japonicum
The genomes of S. chlorophenolicum and S. japonicum are
of similar size (4.57 and 4.46 Mbp, respectively) and contain
a similar number of open reading frames (ORFs) (4,159 and
4,460, respectively). Both S. chlorophenolicum and S. japo-
nicum (Nagata et al. 2010) have a primary chromosome
containing most of the genes for core processes (including
glycolysis, the TCA cycle, amino acid and nucleotide biosyn-
thesis, fatty acid oxidation, DNA replication, transcription,
and translation) and a secondary chromosome. Sphin-
gobiumchlorophenolicumhasasingleplasmid(pSphCh01),
whereas S. japonicum has three (pUT1, pUT2, and pCHQ1).
The third circle in each chromosome map in figure 2
shows in green the positions of genes that encode proteins
in S. chlorophenolicum that have close homologs in S. japo-
nicum that exhibit .80% identity over .90% of the length
of the S. chlorophenolicum sequence. A total of 2,324 S.
chlorophenolicum genes (1,931 on chromosome 1, 285
on chromosome 2, and 108 on pSphCh01) have close ho-
mologs in S. japonicum (see supplementary table 2, Supple-
mentary Material Online). In both chromosomes, regions
with close homologs in S. japonicum show a typical GC
FIG. 3.—Distribution of genes between chromosomes 1 and 2 of
Sphingobium chlorophenolicum. Black bars, fraction of all genes
(excluding those on pSPHCH01); gray bars; fraction of core genes.
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