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Journal of Bacteriology, September 2001, p. 5074-5081, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5074-5081.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic and Structural Organization of the Aminophenol Catabolic
Operon and Its Implication for Evolutionary Process
Hee-Sung
Park and
Hak-Sung
Kim*
Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, 373-1, Kusong-dong,
Yusong-gu, Taejon, 305-701, Korea
Received 3 January 2001/Accepted 29 May 2001
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ABSTRACT |
The aminophenol (AP) catabolic operon in Pseudomonas
putida HS12 mineralizing nitrobenzene was found to contain all
the enzymes responsible for the conversion of AP to pyruvate and acetyl
coenzyme A via extradiol meta cleavage of 2-aminophenol.
The sequence and functional analyses of the corresponding genes of
the operon revealed that the AP catabolic operon
consists of one regulatory gene, nbzR, and the following
nine structural genes, nbzJCaCbDGFEIH, which encode
catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine
zipper motif and a MarR homologous domain. It was also found that
NbzR functions as a repressor for the AP catabolic operon
through binding to the promoter region of the gene cluster in
its dimeric form. A comparative study of the AP catabolic
operon with other meta cleavage operons
led us to suggest that the regulatory unit (nbzR) was
derived from the MarR family and that the structural unit
(nbzJCaCbDGFEIH) has evolved from the ancestral
meta cleavage gene cluster. It is also proposed
that these two functional units assembled through a modular type gene
transfer and then have evolved divergently to
acquire specialized substrate specificities (NbzCaCb and NbzD) and
catalytic function (NbzE), resulting in the creation of the AP
catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type
gene transfer.
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INTRODUCTION |
Nitroaromatic compounds are widely
used in the manufacture of dyes, drugs, explosives, and solvents, and
massive amounts of their discharge have had detrimental effects on the
environment. Much attention has been paid to the bioremediation of
these compounds, and both aerobic and anaerobic degradation by
microorganisms have been reported (13, 16, 45). As a
typical recalcitrant nitroaromatic compound, nitrobenzene (NB) is known
to be metabolized by aerobic bacteria through either an oxidative
pathway (35) or a partial reductive pathway (20, 22,
34, 38). In the partial reductive pathway, NB is converted into
2-aminophenol (AP), which is subsequently metabolized via a
meta-like cleavage pathway. Several enzymes involved in NB
catabolism of Pseudomonas pseudoalcaligenes JS45 were
purified and characterized (21, 23, 24, 31, 44). However,
despite intensive studies on the catabolic pathway of NB and
characterization of the relevant enzymes, a detailed molecular basis of
and a regulatory mechanism for NB catabolism remain yet to be
elucidated. Recently, we reported the novel genetic organization of the
NB catabolic gene clusters that are on the catabolic plasmids pNB1 and
pNB2 in Pseudomonas putida HS12 (37). All the
genes except for that of mutase, which was found on pNB2, were
clustered on pNB1. Of the nbz (for nitrobenzene degradation)
genes, the AP gene cluster was revealed to be a tightly regulated one.
It has been generally accepted that horizontal gene transfer (HGT) has
played an integral role in the dissemination of antibiotic resistance
genes, biodegradative genes, and pathogenicity-conferring genes in
bacteria (7, 10, 27, 32, 36). Recently, it was reported
that HGT is also responsible for bacterial speciation (11,
29). HGT is known to be mediated dominantly by three mobile
genetic elements: conjugative plasmids (for cell contact-dependent gene
transfer), transposons (for Tn-mediated gene transfer), and bacteriophages (which act as the vector for gene transfer) (2, 10, 11). In the gene transfer event, most of mobilized genes cannot be stably maintained in the recipient cells without appropriate selection. Moreover, in order for the transferred genes to confer stable phenotypes (antibiotic resistance, catabolic capability, and
virulence), they have to be assembled into a single functional unit;
otherwise, the whole functional operon needs to be transferred (29). Lawrence and Roth recently proposed the selfish
operon model, in which the gradual formation of gene cluster
and their subsequent integration into an operon is facilitated
by HGT (30). In this way, HGT has played a major role in
the diversification and evolution of microorganisms.
In this paper, to further elucidate the genetic origin and evolutionary
process of the AP catabolic operon, we carried out a genetic
and biochemical characterization of the AP catabolic operon and
investigated its regulatory mechanism. A comparative study of the AP
gene cluster with other well-studied meta cleavage operons was also conducted. Based on the results, we propose
that the AP operon has evolved through modular type gene
transfer, a specific type of HGT. The modular type gene transfer, in
which functional gene units (regulatory and structural gene clusters originated from different sources), not arbitrary genes, are
transferred and assembled into a new operon with specific
function, is considered a fast and efficient mechanism for bacteria to
acquire genetic diversity and expand their metabolic capabilities.
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MATERIALS AND METHODS |
Strains, plasmids, and culture conditions.
Cells of P. putida strains were grown in defined mineral medium at 30°C as
described elsewhere (37). Cells of Escherichia coli strains were cultured in Luria-Bertani medium at 37°C.
pBluescript SK/KS (Stratagene) and pUCP22 (a gift from G. J. Zylstra; GenBank accession no. U07166) vectors were used as cloning
vehicles for E. coli and P. putida
strains, respectively. When necessary, 100 µg of ampicillin/ml was
added to E. coli cultures and 40 µg of gentamicin/ml was
used for P. putida cultures.
DNA manipulation and sequencing.
DNA manipulations, such as
subcloning and transformation, were performed in accordance with the
standard procedure described by Sambrook et al. (41).
Plasmids of P. putida HS12 were isolated as previously
described (37). DNA fragments from pNB1 and pNB2 were
cloned into pBluescript SK/KS and pUCP22, a broad-host-range vector for
gram-negative bacteria. Expression profiles of the constructed plasmid
in E. coli JM109 and P. putida strains were analyzed by assaying the corresponding enzymes.
For DNA sequencing, physical mapping of pNB1 was followed by subcloning
into pBluescript SK/KS. Both strands of the 11.1-kb DNA fragment
from pNB1 were sequenced by the dideoxy-chain termination method,
using double-stranded DNA as the template (42). Sequencing was carried out by a combination of manual sequencing and automated sequencing with an API 377 sequencer (Applied Biosystems Inc.).
Enzyme assays.
Cell extracts were prepared as previously
described (37). The analysis of AP dioxygenase,
2-aminomuconic semialdehyde (AMS) dehydrogenase, and 2-aminomuconate
(AM) deaminase activities was performed by using previously determined
methods (37). 4-Oxalocrotonate (OC) decarboxylase activity
was analyzed spectrophotometrically by measuring the decrease of OC at
236 nm (18). 2-Oxopent-4-enoate (OP) hydratase activity
was determined by the method described by Collinsworth et al.
(8), with some modifications. One unit of activity was
defined as the amount of enzyme required to cause a decrease in
A265 of 1.0 per minute.
4-Hydroxy-2-oxovalerate (HOV) aldolase was assayed by measuring the
oxidation of NADH at A340 in the
presence of excess L-lactate dehydrogenase
(40). To measure acetaldehyde (ACT) dehydrogenase
activity, the coenzyme A-stimulated reduction of
NAD+ was monitored at 340 nm (40).
Chemicals.
2-Hydroxymuconic semialdehyde (HMS) and AM for
AMS dehydrogenase and AM deaminase were synthesized and purified by
using established methods (37). OC was synthesized as
described elsewhere (28, 53). OP was prepared
enzymatically from L-allylglycine as reported by
Collinsworth et al. (8). HOV was prepared by mild alkaline hydrolysis of 4-methyl-2-oxobutyrolactone (40).
RT-PCR.
Total RNA was isolated from HS12 cells grown on NB
or succinate by using an RNeasy total RNA kit (Qiagen). Purified RNA
was treated with RNase-free DNase (Promega) to remove DNA contaminants and then was concentrated by ethanol precipitation. Reverse
transcription (RT)-PCR was carried out with an Access RT-PCR kit
(Promega). The following primer pairs were utilized: P2281 and P3721R
for the nbzCaCbD region, P4801 and P6001R for the
nbzDGF region, and P6361 and P8281R for the
nbzFEIH region (Fig. 1A). The
sequences of the primers are available through personal communication.
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FIG. 1.
(A) The genetic organization of the AP catabolic
operon on the pNB1 plasmid in P. putida HS12.
Arrows indicate the direction of transcription. Duplicated DNA regions
are shown above the physical map. DNA fragments subcloned for
functional analysis are indicated by thin lines below the map. The
locations of the primers and the amplified DNA fragments for RT-PCR are
shown by thick lines. (B) RT-PCR amplification of the AP gene clusters.
Lanes 1, 2, and 3, RT-PCR products with total RNA from NB-grown HS12;
lanes 4, 5, and 6, total RNA from succinate-grown HS12; lanes 7, 8, and
9, PCR products with total RNA from NB-grown HS12 without RT.
Amplifications were performed with the RT1 set (lanes 1, 4, and 7) the
RT2 set (lanes 2, 5, and 8), and the RT3 set (lanes 3, 6, and 9).
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Purification of NbzR and DNA binding assay.
The
nbzR gene was amplified using PCR with a PN primer for the
N-terminal region and a PC primer for the C-terminal region. The
amplified DNA fragment was digested with EcoRI (partial) and HindIII and then was inserted into pMAL-c2X (New England
BioLabs). The recombinant plasmid, designated pMR1, was expressed in
E. coli BL21(DE3) cells under the control of
isopropyl-
-D-thiogalactopyranoside (IPTG). The
overexpressed NbzR protein was cleaved from the maltose binding protein
and purified according to the manufacturer's instructions.
DNA binding experiments were performed by Fried and Crothers' method
(
14), with slight modifications. Two sets of primers,
P793-P1157R and P1300-P1583, were used to amplify the intergenic
regions of
nbzR-
nbzJ and
nbzJ-
nbzCa, respectively. The PCR-amplified
PI
(365 bp from the P793-P1157R set) and PII (284 bp from the
P1300-P1583
set) fragments (50 ng) were incubated with purified
NbzR protein (0 to
300 ng) in 20 µl of binding buffer (10 mM Tris-HCl
[pH 7.2], 10 mM
-mercaptoethanol, 1 mM EDTA, 0.1% Triton X-100,
4% glycerol, 50 mM
KCl, 5 mM MgCl
2) for 10 min at 4°C. The
reaction
mixture was subjected to electrophoresis on 5% native
polyacrylamide
gels in TBE buffer (
41).
Cross-linking experiments.
Cross-linking of NbzR was carried
out as follows. Purified NbzR (1 mg/ml) was first activated by adding 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mM
N-hydroxysuccinimide (NHS) in 20 µl of 10 mM MES (morpholineethanesulfonic acid) (pH 6.0), and then -mercaptoethanol (final concentration, 20 mM) and 20 µl of 50 mM HEPES (pH 8.0) were
added to quench the EDC and raise the pH to 7.5. After incubation at
4°C for 1 h, the reaction was stopped by adding hydroxylamine (final concentration, 10 mM) and analyzed with sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (41).
Nucleotide sequence accession number.
The nucleotide
sequence reported in this study has been deposited in GenBank under the
accession no. AF319593.
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RESULTS AND DISCUSSION |
Sequence and functional analysis of the AP catabolic gene
cluster.
We previously found that the AP catabolic gene cluster
might constitute a single operonic structure in P. putida HS12 (37). To further characterize the AP
catabolic genes, the nucleotide sequence of a 11.1-kb DNA fragment of
the pNB1 plasmid was determined. Analysis of the sequence identified 10 open reading frames (ORFs), all of which were organized in the same
direction with the exception of the nbzR gene, a putative
regulatory gene (Fig. 1A). Databases in GenBank were searched for
proteins having a high degree of similarity to the deduced amino acid
sequences of the AP catabolic gene products by using the BLAST program.
Sequences were then retrieved and compared with the deduced amino acid
sequences by using the ClustalX program. The location of each gene and
the predicted molecular masses of the gene products are listed in Table
1, along with details of proteins bearing
significant similarity to the predicted gene products.
The deduced amino acid sequences of NbzCa, NbzCb, NbzD, and NbzE were
found to have extremely high degrees of identity (98
to 99%) with the
-subunit and
-subunit of AP dioxygenase, the
N-terminal region of
AMS dehydrogenase (residues 1 to 325), and
a partial peptide
sequence of AM deaminase (residues 1 to 54)
from
P. pseudoalcaligenes JS45 (GenBank accession no.
U93363 and
P81593).
Despite the fact that HS12 and JS45 were separately
isolated from
geographically remote areas (
34,
38), DNA and
amino acid
analyses of the above enzymes imply that these strains
are closely
related to each other. Recently, Takenaka et al. reported
the
nucleotide sequence of the AP catabolic genes in
Pseudomonas sp. strain AP-3 (
49). Interestingly, AP structural gene
products
from
Pseudomonas sp. strain AP-3 revealed high
identity levels
with those from HS12, as shown in Table
1.
Pseudomonas sp. strain
AP-3 was isolated on the basis of
growth ability on AP, but HS12
uses NB, not AP, as a growth substrate
and inducer (
37) and
contains all the catabolic genes for
NB degradation. Nonetheless,
AP gene clusters in AP-3 and HS12 seem to
have a similar physiological
role in AP catabolism. NbzJ has 20% amino
acid identity with chloroplast-type
ferredoxin (XylT) in the TOL
operon that mediates the reductive
reactivation of catechol
2,3-dioxygenase (
25). Lendenmann and
Spain reported that
purified AP dioxygenase from JS45 was severely
inactivated by catechol
(
31), as was found to be the case for
the inactivation of
catechol 2,3-dioxgenase by 4-methylcatechol
(
25). This
suggests the possible participation of NbzJ in the
protection and
reactivation of NbzCaCb. However, because only
two of four cystein
residues presumed to serve as ligands for
the [2Fe-2S] cluster in
XylT were conserved in NbzJ (data not
shown), it is not clear yet
whether NbzJ is involved in AP catabolism.
When compared with other
meta cleavage gene clusters, NbzD showed
55% amino acid
identity with HMS dehydrogenase (XylG) from
P. putida mt-2,
but NbzCa and NbzCb had relatively low identities
with other extradiol
dioxygenases, such as HpcB from
Escherichia coli and CarBb
from
Pseudomonas sp. (Table
1). As for NbzE, no
significant
level of similarity with other deaminases has been
found. Instead, part
of NbzE (residues 44 to 111) possessed an
identity of 25% with
4-oxalocrotonate tautomerase (XylH) from
P. putida mt-2
(Table
1). Interestingly, the gene products of
nbzF, nbzG,
nbzH, and
nbzI exhibited relatively high levels of
identity with the well-characterized
meta cleavage enzymes
(XylI,
XylJ, XylK, and XylQ) of
P. putida mt-2 (Table
1). In
order to
confirm the predicted catalytic function of the AP catabolic
gene
products, a functional analysis of the subclones containing the
ORFs was attempted with
E. coli JM109 cells (Fig.
1A and
Table
2). All the subclones carrying part
or all of the catabolic genes
exhibited the corresponding catalytic
activities, as predicted
by sequence comparison analysis. Based on the
above results, the
AP catabolic gene products are as follows: NbzCaCb,
- and
-subunits
of AP dioxygenase; NbzD, AMS dehydrogenase; NbzE,
AM deaminase;
NbzF, OC decarboxylase; NbzG, OP hydratase; NbzH, HOV
aldolase;
and NbzI, ACT dehydrogenase.
In order to determine whether the AP catabolic genes are cotranscribed,
three sets of primers were designed (Fig.
1A) and
used for PCR
amplification with total RNAs from NB-grown and succinate-grown
P. putida HS12. The RT1 set (spanning the
nbzCaCbD gene region),
RT2 set (spanning the
nbzDGF gene region), and RT3 set (spanning
the
nbzFEIH gene region) resulted in the amplification of 1.4-,
1.2-, and 1.9-kb DNA fragments, respectively, only from a
reverse-transcribed
RNA sample of HS12 grown on NB (Fig.
1B). This
result indicates
that the
nbzCaCbDGFEIH genes are
transcribed as a single transcript
and their expressions are
coordinately regulated as a whole, thus
confirming the conclusion that
these genes constitute a single
operon. Although Takenaka
et al. reported a structural gene organization
of
amn genes
(
amnBACFDEHG) that is identical to that of
nbz
genes
of the AP operon (
49), all the structural
gene products are
not functionally identified, and whether these genes
are clustered
in a single operon was not investigated. ORF1 in
P. pseudoalcaligenes JS45 was classified as a putative
member of the YjgF protein family
and found to be cotranscribed with
the
amnBAC genes (
9). High
DNA identity (98%)
between the
NbzJ-
nbzCaCb region in HS12 and
the
orf1-
amnBA region in JS45 implies that, as in
JS45,
NbzJ in
the AP gene cluster of HS12 might be
cotranscribed with the other
structural genes,
nbzCaCbDGFEIH. Therefore, the overall genetic
organization
of the AP catabolic gene cluster is probably
nbzJCaCbDGFEIH.
A truncated duplicate of
nbzDG genes was found in the downstream
of the structural
genes. But, the possible involvement of this
region in the catabolism
is not clear
yet.
Regulation of the AP operon and NbzR.
The catabolic
genes and their products of the AP operon were studied
extensively (21, 23, 31, 37, 48), but the regulatory mechanism of the operon has not been reported so far. It was
previously observed that the AP-degrading pathway is inducible
(37). When pUP3951, containing only the nbzB
gene (1.0 kb) from the pNB2 plasmid, was expressed in P. putida HS124 harboring a pNB1 plasmid with the nbzA and
nbzJCaCbDGFEIH genes, the induction profile of the AP
catabolic genes and the ability of P. putida HS124 (pNB1 + pUP3951) to grow on NB were similar to those of the wild-type HS12
(pNB1 + pNB2) (Fig. 2B). Only NbzB from
pNB2 is required both for the growth of HS12 on NB and for the full
induction of the AP catabolic genes by NB. Therefore, contrary to a
previous report (37) suggesting the possible existence of
a regulatory factor in pNB2, we reasoned that a potential regulatory
factor(s) controlling the expression of the AP catabolic genes might be located on the pNB1 plasmid based on the above results. The presence of
mutase from pNB2 only contributes to the conversion of
hydroxylaminobenzene to AP (37). To get some insights into
the regulatory mechanism of the AP operon, several plasmids
were constructed, and the expression profile was analyzed by assaying
AP dioxygenase activity in HS120, a derivative of HS12 cured of pNB1
and pNB2. As shown in Fig. 2, unlike deletion of both the
nbzR gene and putative promoter region on the AP
operon in HS120 (pUP42), only deletion of the nbzR
gene in HS120 (pUP41) led to the constitutive expression of the
AP-encoded pathway regardless of the growth substrates utilized. This
result indicates that NbzR is a negative regulator and that the
promoter of the AP operon is located upstream of the
nbzJ gene. Although only NB was found to be an inducer for the AP catabolic operon in wild-type P. putida
HS12 (pNB1 plus pNB2) and P. putida HS124 (pNB1
plus pUP3951), NB could not induce the expression of the AP
operon significantly in P. putida HS120 (pUP40 or
pUP50) containing only the nbzR-NbzJCaCb or the
nbzR-NbzJCaCbDGFE region, which suggests the requirement of
secondary trans-acting factor(s) for the regular induction
of the AP operon.
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FIG. 2.
(A) Construction of plasmids containing various regions
of the AP operon. DNA fragments used for subcloning are
indicated by open bars below the physical map. (B) Expression profile
of the AP dioxygenase from strains with different plasmids. The carbon
source used as a growth substrate or an inducer is indicated in
parentheses (NB, nitrobenzene; AP, 2-aminophenol; suc, succinate). 12, HS12; 124, HS124; 120, HS120. Plasmid pUP3951 was constructed by
cloning a 1.0-kb SalI-SphI DNA fragment
(the nbzB gene) from pNB2 (37) into
pUCP22.
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To determine whether the putative regulator NbzR is involved in the
expression of the AP operon through direct contact with
the
promoter region, a gel mobility shift assay with the purified
NbzR and
the presumed promoter region was carried out. Figure
3 shows a clear mobility shift of the PI
fragment containing the
putative promoter region (Pap) of the AP
operon by NbzR, whereas
no retardation was observed with the
PII fragment containing an
intergenic region of the
nbzJ and
the
nbzCa genes. As expected
from the induction profile of
HS120 (pUP40) in Fig.
2B, neither
NB nor AP affected the binding of
NbzR to the promoter region.
From the gel mobility assay (Fig.
3) and
the induction profile
of HS120 (Fig.
2), the NbzR binding site and the
transcription
initiation site of the AP operon were presumed to
be located upstream
of the
nbzJ gene. We carried out a
footprinting analysis and a
primer extension assay to determine the
precise NbzR binding site
and transcription start site, but we could
not identify the exact
sites under our experimental conditions.
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FIG. 3.
Specific binding of NbzR to
nbzR-nbzJ intergenic region. DNA
fragments (PI and PII) used for the binding assay are indicated by bars
below the physical map. For the gel mobility shift assay, various
amounts of purified NbzR (0 to 300 ng) were incubated with a PI or PII
DNA fragment. NB or AP was added optionally at a final concentration of
10 mM. The PI-NbzR complex, PI, and PII are indicated by arrows.
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The C-terminal region of the predicted NbzR (residues 77 to 193) shared
a modest identity with a group of negative transcription
regulators
belonging to the MarR family (
47). In addition, NbzR
possessed a putative basic leucine zipper motif with a basic region
and
an imperfect heptad leucine repeat (
6) in the N-terminal
region (residues 34 to 73), which is expected to be responsible
for the
dimerization and DNA binding of NbzR (Fig.
4A and B).
Based on these observations,
it is concluded that the NbzR protein
acts as a dimeric repressor for
the AP catabolic operon through
specific binding to the
divergently located promoter regions of
the
nbzR gene and
the AP structural genes.
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FIG. 4.
(A) Schematic domain structure of NbzR. The typical
characteristics of bZIP, repeating leucine residues and a preceding
basic region, and an alignment of NbzR (residues 77 to 193) with MarR
from E. coli K-12 (GenBank accession no. AE000250) are
shown below. A preliminary consensus sequence (DXRXXXXXLTXXG) of
the MarR family (47) is shown above the alignment. The
putative HTH regions in MarR are indicated below the alignment. Amino
acids highlighted in black boxes are identical residues. #, Residues of
MarR causing negative complementary trans-dominant
mutations; *, residue of MarR making a specific operator contact. (B)
Chemical cross-linking of NbzR by EDC. NbzR (1 mg/ml) was reacted with
2 mM EDC and 5 mM NHS at 4°C for different times as described in
Materials and Methods. Arrows indicate monomeric and dimeric forms of
NbzR.
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Comparative study of AP operon and its origin.
A
comparative study of the catabolic pathway and the genetic organization
of the AP operon with other well-studied meta
cleavage gene clusters provides an intriguing insight into the
evolution of the AP catabolic operon. In general, microbial
catabolic routes of various aromatic compounds converge at the ring
cleavage steps, that is, at the ortho and meta
cleavage pathways which are responsible for the conversion of catechol
derivatives into Krebs cycle intermediates, such as pyruvate and acetyl
coenzyme A (52). The NB catabolic pathway also follows
this general route. NB is first transformed to AP, a catechol analogue,
by a partial reductive pathway involving NB nitroreductase (NbzA) and
HAB mutase (NbzB), and then AP is further metabolized via a
meta-like cleavage pathway.
In most of the
meta cleavage pathways, two main different
catabolic branches exist, the oxalocrotonate pathway (central
meta pathway) and the hydrolytic route, which is signified
by the presence
of hydrolase (Fig.
5).
These two branches have different substrate
preferences in the TOL
pathway. Catechol and 4-methylcatechol
are catabolized via the
oxalocrotonate pathway, but 3-methylcatechol
is dissimilated via the
hydrolytic branch (
19). The AP catabolic
pathway has a
distinct ring cleavage pathway as depicted in Fig.
5. It has only the
central
meta cleavage pathway and unique catabolic
enzymes
that differentiate the AP catabolic pathway from common
meta
cleavage pathways, especially when it comes to ring cleavage
(AP
dioxygenase), dehydration (AMS dehydrogenase), and deamination
(AM
deaminase) reactions. It has been proposed that the hydrolytic
branch
in the
meta cleavage pathway has been recruited from
cellular
hydrolase to degrade keto ring products (
26). The
absence of
hydrolase in the AP catabolic operon suggests that
the recruitment
event of the AP structural genes may have preceded the
incorporation
of hydrolytic branch in ancestral
meta
cleavage operon (see below).
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FIG. 5.
Comparison of the AP catabolic pathway with other
meta cleavage pathways encoded on the TOL, NAH, and DMP
operons.
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A widespread distribution of common
meta cleavage routes in
different catabolic pathways provides a typical clue to horizontal
transfer of catabolic gene clusters in the course of adaptation
to
organic compounds available in the natural environment
(
52).
The well-conserved genetic organization of the
meta cleavage pathway
has been reported for the toluene
degradation pathway encoded
in the TOL plasmid of
P. putida
mt-2 (
17), the dimethylphenol
operon on plasmid
pVI150 of
Pseudomonas sp. CF600 (
43), and
the
naphthalene operon on the chromosome of
Pseudomonas
stutzeri AN10 (
4). Comparison of the AP catabolic
operon with the
meta cleavage operons
revealed some interesting facts. While the gene
products of
nbzF,
nbzG, nbzH, and
nbzI, which encode the same
catabolic
enzymes of the
meta cleavage operons, showed
relatively
high amino acid identities (40 to 78%), the gene
products of
nbzCaCb and
nbzE, which encode
enzymes with different specificities and
distinctive catalytic
functions, shared low levels of identity
with the counterparts of
meta cleavage operons (15 to 25%).
NbzCaCb, as in the case of AmnBA in
Pseudomonas sp. strain
AP-3 (
48) and
P. pseudoalcaligenes JS45
(
9), consists of a
-subunit containing conserved
histidine residues (His-14, His-65,
His-141, His-205), which seem to
constitute a heme iron cofactor
coordinate site found in class III
extradiol ring cleavage enzymes
proposed by Spence et al.
(
46), and an
-subunit possibly contributing
to the
stabilization of the heterotetrameric structure (
48).
High
substrate specificity towards AP, rather than catechol
(
31),
as well as the above result support the hypothesis
that NbzCaCb
would have divergently evolved from an ancestral gene for
class
III ring cleavage enzymes through a gene duplication mechanism
(
50) to acquire the specific ring cleavage function toward
AP.
Despite a high degree of identity between NbzD and HMS
dehydrogenase
of the
meta cleavage pathway and the
well-conserved putative NAD
+ binding site in NbzD
(data not shown), the substrate binding
affinity of NbzD towards AMS is
three times higher than that of
HMS (
23), which also
implies the occurrence of divergent evolution
of NbzD from HMS
dehydrogenase of the ancestral
meta cleavage
pathway. AM
deaminase, which is a unique enzyme found only in
the AP catabolic
pathway, was reported to have biochemical characteristics
similar to
those of 4-oxalocrotonate tautomerase (XylH) from the
TOL plasmid. Both
of these enzymes have hexameric structures and
share tautomerization
reactions in catalysis (
21). Analyzing
the amino acid
sequence of NbzE, we could not find any similarity
with other
deaminases reported so far, and NbzE seems not to possess
any cofactor
binding site commonly found in several types of deamination-catalyzing
enzymes, such as PLP in aminotransferase (
12),
NAD
+ in glutamate dehydrogenase
(
51), and a Zn
+ coordinate site in
adenosine deaminases (
3). Instead, we observed
a modest
level of amino acid identity between part of NbzE and
XylH, and the
nbzE gene has identity as high as 54% with the
xylH gene, which implies a close evolutionary relationship
between
these genes. Based on the above observations, it is proposed
that
NbzE might have evolved from 4-oxalocrotonate tautomerase of the
ancestral
meta cleavage operon to transform an amino
group to
a keto one, leading to the subsequent degradation in the
meta cleavage pathway. Further biochemical study including
site-directed
mutagenesis would be necessary to elucidate a more
detailed catalytic
mechanism and the genetic origin of AM deaminase.
Thus, it can
be concluded that the structural genes of the AP
operon might
have originated from ancestral
meta
cleavage gene cluster and
have divergently evolved for the catabolism
of
AP.
The AP operon has a different regulatory system from that of
the
meta cleavage operons that are regulated by one
of the following
regulatory systems: LysR (
52), AraC/XylS
(
15), and a
54-dependent
regulator (
5). NbzR, which acts as a repressor for
the
expression of the AP operon, contains two distinct motifs,
a
putative basic leucine zipper motif and a MarR homologous domain.
Recently, it was shown that the MarR protein has two helix-turn-helix
(HTH) DNA binding motifs (
1), as shown in Fig.
4A.
Although
the MarR homologous domain contains the consensus sequence
(DXRXXXXLTXXG)
as proposed by Sulavik et al. (
47) and some
conserved sequences
in HTH regions, many of the residues (Glu-69,
Ala-70, The-72,
Arg-73, Gln-110, and Gly-116) critical for DNA binding
were not
conserved in NbzR. In particular, Arg-73, which makes a
specific
contact with the operator sequence, was replaced with Glu, a
negatively
charged amino acid. In addition to the incongruity in
critical
amino acid residues in HTH motifs and operator DNA sequences
(
1)
(Fig.
3B), NbzR was observed to bind to the promoter
region probably
as a dimer, in contrast to oligomeric binding of the
MarR protein
(
33). Many of the proteins belonging to the
MarR family are
known to interact with phenolic compounds, whereas NbzR
did not
interact with AP, as observed in Fig.
2 and
3. These results
suggest
that the MarR homologous domain of NbzR do not contribute to
DNA
binding, and a putative basic leucine zipper motif in the
N-terminal
region may lead to a dimerization and the consequent DNA
binding
of NbzR to the Pap promoter region. The elucidation of
derepression
and the DNA binding mechanism of NbzR in vivo should
provide a
more detailed regulatory system of the AP catabolic
operon. From
the above analyses, it is reasonable to suggest
that the regulatory
unit in the AP operon might have originated
from the Mar gene
cluster and has become involved in the
regulation of the AP structural
gene
expression.
Evolution of AP operon in a modular fashion.
Based on
previous results (37) and a comparative study of the AP
catabolic genes, it is inferred that P. putida HS12 has acquired nbzA, nbzB, and the AP catabolic
operon from different sources in order to adapt to an
NB-contaminated habitat. Unlike nbzA and nbzB
genes, which are scattered and free of regulation to some extent, the
AP catabolic gene cluster was revealed to form a well-defined
operonic structure under tight regulation. Thus, the
evolutionary process of the AP operon is expected to provide a
critical insight into the gene transfer mechanism. In general, HGT in
bacteria is mediated by transformation, conjugation, and transduction
(2, 10, 11). It has been known that bacterial evolution
towards the acquisition of antibiotic resistance and pathogenicity-conferring genes, expansion of xenobiotics degradation capabilities, and speciation has proceeded via the horizontal transfer
of gene(s) or whole operon, followed by modification of the
respective gene(s) (7, 10, 27, 32, 36). However, it is
unlikely that the HGT model can fully explain the mechanism by which
bacteria have efficiently acquired genetic diversity and metabolic
capabilities. In this regard, the present study on the organization and
origin of the regulatory and structural units of the AP catabolic
operon led us to propose that the AP catabolic operon
has evolved in a modular fashion. Herein, we term it modular type gene
transfer, which refers to a specific kind of HGT comprising fusion of
functional gene units (regulatory and structural gene clusters)
originating from different sources and their subsequent arrangement,
resulting in the rapid generation of genetic diversity in bacteria. In
the creation of the AP operon on plasmid pNB1, both the
regulatory unit (nbzR), which seems to have been derived
from the Mar operon, and the structural unit (nbzJCaCbDFGEIH), from the ancestral meta
cleavage operon, assembled through modular type gene transfer
and then rearranged for adaptation, as depicted in Fig
6. HGT through a conjugative catabolic
plasmid to indigenous bacteria for efficient xenobiotics utilization
was observed in the natural environment (39), and we also
found a Tn5501-like transposon in the catabolic plasmid of
HS12 (37). Maybe the mobile genetic elements, such as
conjugative plasmids and transposons, might have been involved in the
gene transfer. During the fusion of gene clusters into an
operon, directional mutations, such as gene rearrangement
(differential genetic organization of the structural genes;
nbzJCaCbDGFEIH), gene duplication (truncated genes found
downstream of the operon; nbzDG gene segment), gene incorporation (DNA binding domain of bZIP; nbzR), and point
mutations (generation of new catalytic functions; nbzC,
nbzD, and nbzE), must have occurred for the
creation of the AP operon. Therefore, the evolutionary process
of the AP operon provides evidence that modular type gene
transfer is considered an efficient mechanism for bacteria to create
genetic diversities and to expand metabolic capabilities.
View larger version (25K):
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|
FIG. 6.
Proposed mechanism for the creation of the AP
operon. Modular type gene transfer of regulatory and structural
units was followed by fusion of these gene clusters into an
operon. Boldtype ORFs in the AP operon represent
enzymes with different substrate specificities and catalytic function
from meta cleavage operons.
|
|
| |
ACKNOWLEDGMENTS |
We are grateful to J. C. Spain, P. R. Reeves, J. Davison, A. M. Campbell, and A. Salyers for critical comments on
the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Korea Advanced Institute of Science and
Technology, 373-1, Kusong-dong, Yusong-gu, Taejon, 305-701, Korea.
Phone: 82-42-869-2616. Fax: 82-42-869-2610. E-mail:
hskim{at}mail.kaist.ac.kr.
| |
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Journal of Bacteriology, September 2001, p. 5074-5081, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5074-5081.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
