FEMS Microbiology Reviews 15 (1994) 239-249

g' 1994 Federation of European Microbiological Societies 0168-6445/94/$15.0t)

Published by Elsevier
239
FEMSRE 00433
Genetic adaptation of bacteria to chlorinated
aromatic compounds
J a n Ro e l o f v a n d e r Me e r *
EAWAG, Ueberlandstr. 133, Ctt-8600 Diibendo( Switzerland
Abstracc" Genetic mechanisms in bacteria provide a continuous source of aherations in DNA sequences that may lead to
favourable adaptations. Bacteria that use chlorinated aromatics as sole carbon and energy sources show evidence of these different
genetic alterations. The distinct effects of single base-pair mutations on adaptation of bacterial strains (e.g. by changing the
substrate specificity of a key metabolic enzyme or regulator protein) have been demonstrated in various studies. In addition to
these small sequence modifications, intermolecular or intercellular gene exchange mechanisms can result in new strains with
altered metabolic capabilities. The details of these evolutionary processes with respect to the metabolism of chlorobenzenes and
chlorocatechols are reviewed in this manuscript.
Key words: Adaptation; Pseudomonads; Chlorinated aromatic compounds: Evolution
I n t r o d u c t i o n
Over t he past few decades, t he l arge scale
i ndust ri al pr oduct i on and ext ensi ve use of syn-
t het i c chemi cal s in all levels of soci et y have led to
a wi de di st ri but i on of har mf ul compounds in t he
envi r onment . In al most any c ompa r t me nt of our
envi r onment (e.g. soil, air, wat er , and bi ot a),
t r aces of synt het i c or gani c c ompounds can be
det ect ed. At many wast e di sposal sites or ot her
poi nt sources of cont ami nat i on, concent r at i ons of
t hese chemi cal s may be so gr eat t hat di rect toxi-
col ogi cal risks ar e encount er ed.
Mi cr oor gani sms pl ay a cent r al rol e in t he
degr adat i on of or gani c c ompounds and t her ef or e
ar e very i mpor t ant for t he mi ner al i zat i on or
* Corresponding author. Tel.: ( + 41-1 )823 5438; Fax: ( + 41-1 )
823 5547
det oxi fi cat i on of toxic organi c chemi cal s. Unf or -
t unat el y, not all or gani c compounds arc equal l y
well bi odegr adabl e, and, in some cases, envi ron-
ment al condi t i ons may be unf avour abl e for mi-
crobi al activity. I f t he mol ecul ar st r uct ur e of a
c ompound is not commonl y encount er ed in na-
t ure, mi cr oor gani sms may not have t he necessar y
degr adat i ve enzyme systems. The pr es ence of
subst i t uent s, such as hal ogen at oms, ni t ro- or
sulfite groups, on ot her wi se easily degr adabl e
compounds may make t he compound less accessi-
ble for bi odegr adat i on and t hus mor e per si st ent
in t he envi r onment [1,2].
It has been shown t hat mi crobi al popul at i ons
may adapt to use previ ousl y per si st ent com-
pounds as novel car bon and ener gy sources [3-5].
Such an adapt at i on may be caused by t he sel ec-
t i on of mut ant st rai ns which have acqui r ed novel
met abol i c activities or al t er ed enzymat i c specifici-
ties. Her e 1 discuss some of t he genet i c mecha-
SSDI 0168- 6445( 94) 00035- W
24(I
nisms which may have r esul t ed in t he f or mat i on
of t he met abol i c pat hway of chl or i nat ed benzene
degr adat i on in ps eudomonads .
Genetic mechanisms for adaptation
A wi de vari et y of pr ocesses exist t hat cause
changes in existing genet i c mat er i al and resul t in
al t er ed met abol i c funct i ons. The det ai l s of t he
di f f er ent genet i c mechani s ms in adapt at i on to
xenobi ot i c compounds have recent l y been re-
vi ewed el sewher e [6,7]. When consi der i ng t he dif-
f er ent genet i c mechani s ms involved in evol ut i on
and al t er at i ons of DNA sequences, a di st i nct i on
shoul d be made bet ween vert i cal and hor i zont al
evol ut i onar y pr ocesses [8]. Vert i cal pr ocesses are
t hose which l ead to a di ver gence in DNA se-
quence in daught er cells, due to t he accumul at i on
of mut at i ons. Thes e mut at i ons can be single
bas e- pai r changes or t hose l eadi ng to l arger se-
quence changes, e.g. del et i ons and dupl i cat i ons.
The ef f ect s of poi nt mut at i ons on enzyme speci -
ficity or on ef f ect or r ecogni t i on by r egul at or pr o-
t ei ns have been well est abl i shed [9-12], and it has
be c ome cl ear t hat t hese pr ocesses can have di rect
consequences for t he adapt at i on of st rai ns to
xenobi ot i c compounds . Ot he r possi bl e pr ocesses
of sequence di ver gence, e.g. sl i pped st r and mi s-
pai ri ng, can l ead to mor e ext ensi ve sequence
changes and may be a gener al driving force for
t he di ver gence of gene sequences [13-15]. The
effect of such a pr ocess in mi cr oor gani sms, how-
ever, has not yet been well i nvest i gat ed.
Hor i zont al pr ocesses ar e t hose which cause an
exchange of DNA sequences bet ween t he ge nome
of two di f f er ent or gani sms ( i nt er cel l ul ar move-
merit), or bet ween di f f er ent DNA mol ecul es, e.g.
wi t hi n t he c hr omos ome and ext r achr omos omal
el ement s inside one or gani sm ( i nt er mol ecul ar
move me nt ) [8]. Hor i zont al move me nt of genet i c
i nf or mat i on may be caused by r ecombi nat i on or
ot her mechani s ms of gene exchange, e.g. conj uga-
tion, t r ansduct i on, or t r ansf or mat i on. It has be-
come very cl ear t hat hor i zont al gene exchange
pl ays an i mpor t ant rol e in t he adapt at i on of mi -
cr oor gani sms to xenobi ot i c compounds [6]. The
list of bact er i a wi t h sel f - t r ansmi ssabl c pl asmi ds
encodi ng t he degr adat i on of ar omat i c or toxic
or gani c compounds is ext ensi ve [16]. Al t hough
many of t hese pl asmi ds have a si mi l ar backbone
st r uct ur e encodi ng pr obabl y repl i cat i on and
t r ansf er funct i ons, t hey carry di f f er ent cat abol i c
genes [17]. Thi s st rongl y suggest s t he exi st encc of
pr ocesses by which pl asmi ds can acqui re genes or
gene cl ust ers and subsequent l y di ssemi nat e t hem
in a mi crobi al popul at i on [18,19]. Such a r ecent
exampl e of hor i zont al gene exchange has been
t he di scovery of t r ansposabl e el ement s of differ-
ent fami l i cs cont ai ni ng cat abol i c genes. The xyl
and nah genes arc l ocat ed on Tn3- t ype t rans-
posons, Tn4561 (56 kb) [2(I] and Tn4655 (55 kb)
[21], respect i vel y. Ot he r el ement s whi ch have been
descr i bed i ncl ude Tn5271, encodi ng 4- chl or oben-
zoat e met abol i sm [22], Tn4371, for 4-chl orobi -
phenyl met abol i sm [23], 1S931, associ at ed with
t he 2, 4, 5-t ri chl orophenoxyacet i c acid genes [24],
the DEH el ement , which carri es t he dehal oge-
nase genes of Pseudomonas putida PP3 [25], and
Tn5280 of t he chl or obenzene pl asmi d pP51 [26]
(see below). Some of t hese el ement s resi de on t he
chr omos ome (such as IS931, Tn4371, and DEH),
wher eas t he ot her s wer e originally i sol at ed from
pl asmi ds.
Evolution of the metabolic pathway for chloro-
benzene degradation
Since very few bact er i a had been descr i bed
which wer e abl e to degr ade chl or obenzenes com-
pletely, we consi der ed t hese compounds sui t abl e
subst r at es for evol ut i onar y st udi es of bact eri al
adapt at i on. Char act er i zat i on of Pseudomonas sp.
st rai n P51, capabl e of degr adi ng 1, 2, 4-trichloro-
benzene, 1, 2-dichloro- and 1, 4-di chl orobenzene,
al ong with st rai ns i sol at ed by ot her s [5, 27-29], led
to a r easonabl y good pi ct ure of t he met abol i c
pat hway for chl or obenzene degr adat i on and of
t he di f f er ent genes which are i nvol ved in this
pat hway [26,29-31]. The first enzymes of the
pat hway, a t hr ee- component ar omat i c ring dioxy-
genase and a benzene glycol dehydr ogenase, cat -
alyze t he i ncor por at i on of a dioxygen mol ecul e
into t he ar omat i c ring and t he subsequent dehy-
dr ogenat i on which gives rise to a chl or i nat ed
catechol. The chlorinated catechol is then cleaved
in a second dioxygenation reaction, catalyzed by
chlorocatechol 1,2-dioxygenase, and furt her de-
graded, via a number of different chlorinated
intermediates, probably to (chloro-)3-oxoadipate.
In strain P51 the different genes encoding these
metabolic steps were found on a l l 0-kb plasmid
[29]. Thr ee different transcriptional units were
241
found: (i) the ' upper pathway' cluster, containing
the genes encoding the chl orobenzene dioxyge-
nase and the chl orobenzene glycol dehydroge-
nase; (ii) the ' chlorocatechol oxidative' cluster,
encoding the genes for the conversion of chloro-
catechols (see below); and (iii) the regulatory
gene tcbR, which encodes a transcriptional acti-
vator for the chlorocatechol oxidative operon
+
v v . , ,~ ~,..<~_.%,
Cl % ' ~ ~ 0 C ~ [ ~
,
H e l ] O H I
I
O H " T C, O H 0 , " " T e l
c t / c l i
12 0 c,
13 n ] c~ ci cl.
. M e i : a . / I , o o o . ~ o o O r t h o
NO COOH HO H H COON
HOOC H ]
H C O O H J
Fig. 1. Schematic represent at i on of metabolic channel i ng in the aerobic degradat i on of aromatic compounds by bacteria. (A) A
number of different aromatic compounds and t he positions at which initial enzymatic attack can take place (indicated by numbered
arrows). Dot t ed arrows between different structures indicate t hat this particular compound can occur as intermediate in the
degradat i on of the previous one. Numbers: 1, biphenyl dioxygenase [82]; 2, dibenzofuran dioxygenase [83]; 3, napht hal enesul foni c
acid dioxygenase [84]; 4, napht hal ene dioxygenase [85,86]; 5, dibenzo-p-dioxin dioxygenase [83]; 6, ' 2,4-dichlorophenoxyacetate
monooxygenase' [69], 2, 4- D/ a- ket ogl ut ar at e dioxygenase [49]; 7, T-HCH dehydrochlorinase [87]; 8, 1,2-dihydroxynaphthalene
dioxygenase [33,84]; 9, 2,2' ,3-trihydroxybiphenyl dioxygenase [56]; 10, pent achl orophenol 4-monooxygenase [88,89]; 11, 2,3-dihy-
droxybiphenyl dioxygenase [90]; 12, benzoat e 1,2-dioxygenase [44]; 13, 4-chlorobenzoate dehalogenase [58]; 14, phenol hydroxylase
[50]: 15, xylene monooxgenase [91]; 16, toluene dioxygenase [32]; 17, toluene 4-monooxygenase [51]; 18, di ni t rot ol uene dioxygenase
[92]; 19, salicylate hydroxylase [47]; 20, benzene sulfonate dioxygenase [93]; 21, chl orobenzene dioxygenase [29]; 22, 2,4-dichlorophe-
nol hydroxylase [38]. Different shadings indicate enzyme families with sequence homologies o1 with comparable activities: (black).
extradiol dioxygenases; (stippled), aromatic ring dioxygenase; (grey dotted), monooxygenases and hydroxylases; (striped), dehaloge-
nase activities. (B) The different ortho dihydroxylated central intermediates. Ring cleavage of these i nt ermedi at es takes place as
indicated by the arrows. Open arrows represent intradiol dioxygenases, filled arrows the extradiol dioxygenases. Numbers: 1,
catechol 2,3-dioxygenase; 2, catechol 1,2-dioxygenase; 3, prot ocat echuat e 4,5-dioxygenase; 4, prot ocat echuat e 3,4-dioxygenase; 5,
chlorocatechol 1,2-dioxygenase. Below this are shown the ring cleavage products of the intradiol- (ortho-) and extradiol- (meta)
cleavage reactions. No complete overview of all possible reactions is intended.
242
[26,29-31]. The upper pat hway gene cluster in
strain P51 is flanked by two iso-insertion ele-
ments, ISI06O and IS1007. This compl et e ele-
ment, Tn5280, was shown to be a functional
transposon, able to insert in single copy and at
random into the genome [26].
As a result of these analyses, it appears that
the met abol i c pathway for chl orobenzene degra-
dation in strain P51 devel oped from two different
genetic elements. The t ransposabl e cl cment con-
taining the genes for the aromat i c ring dioxygc-
nase and the benzene glycol dehydrogenase, i.e.
Tn5280, may havc originated in bacteria degrad-
ing toluene by direct dioxygenation, such as P.
putida F1 [32]. The dioxygenase in this strain was
shown to have a broad substrate range, and could
oxidize chlorinated benzenes to the correspond-
ing dihydrodiols [32]. If such a dioxygenase gene
cluster became capt ured by two copies of an
insertion element, i.e. IS1066 and IS1007, suc-
cessful t ransfcr of the dioxygenase t ransposon to
a catabolic plasmid containing a chlorocatechol
oxidative operon, could provide the resulting
transconj ugant strain with the necessary genetic
information to carry out compl et e chl orobenzene
degradat i on. Strain P51, then, provides a nice
exampl e of different cw)lutionary mechani sms
which may bc required for the generat i on of a
new catabolic pathway, i.e. (i) recombi nat i on
events involving horizontal gene transfer, and (ii)
vertical evolution of specialized enzyme systems
for new (chlorinated) substrates (see below).
Convergence and vari at i ons in met abol i c path-
ways for ( chl oro- ) aromat i c c ompounds
It has been established that met abol i c path-
ways of aromat i c compounds in bact eri a generally
follow similar strategies and involve a limited
number of central steps. Pathways for aerobic
degradat i on of aromatics apparent l y converge to
form a relatively small number of i nt ermedi at es
(Fig. 1). These i nt ermedi at es carry at least two
hydroxyl groups (in ortho or para positions) and
can contain ot her substituent groups. They arc
then cleaved ei t her by intradiol dioxygenase en-
zymes (ortho cleavage) or by extradiol dioxyge-
nases (meta cleavage). The intradiol and extra-
diol dioxygenases appear to have no significant
similarities on the amino acid level, and t herefore
are not evolutionary closely rel at ed [33]. Within
the extradiol dioxygenases and intradiol dioxyge-
nases, different enzyme groups also exist with
relatively little sequence similarity. The archetype
extradiol dioxygenase, catechol 2,3-dioxygenase
(encoded by the xylE, nahH or dmpB genes)
(Fig. IB), does not show significant sequence
similarity with the extradiol enzyme prot ocat e-
chuat e 4,5-dioxygenase [34]. Of the intradiol
dioxygenases, three subgroups have been de-
scribed: catechol 1,2-dioxygenase (encoded by
catA of Acinetobacter calcoaceticus or P. putida )
[14], prot ocat echuat e 3,4-dioxygenase (encoded by
the pcaHG genes of e. g. P, putida, P. cepacia, A.
calcoaceticus) [35 -37], and chlorocatechol 1,2-di-
oxygenase (encoded by tcbC of Pseudomonas sp.
strain P51 [30], tfdC of Alcaligenes eutrophus
[38,39], or ch' A of P. putida pAC27 [40], and
described for Pseudomonas sp. B13 [41]).
Among the enzymes which catalyze the initial
steps in aromat i c degradat i on pathways, different
classes are found with extensive homology to each
ot her and to ot her enzymes of the central path-
ways (Fig. I), suggesting similar evolutionary
strategies. The most i mport ant class of these en-
zymes is probably that of the aromat i c ring dioxy-
genases, which catalyze insertion into the aro-
matic ring of two hydroxyl groups derived from
molecular oxygen and cofactors such as NADH
(recently reviewed in references [42,43]). It cur-
rently appears that all of these dioxygenases are
mul t i -component enzymes with three or four dif-
ferent protein subunits. These proteins comprise
a short electron transfer chain, by which electrons
are t ransferred from NADH, via a reduct ase and
a ferredoxin, to the terminal oxidase. The reduc-
tase and the ferredoxin may be combi ned as two
domai ns of one protein molecule, as is the case
for the toluate and benzoat e dioxygenases [13,44].
In some cases, the different protein subunits of
the aromat i c ring dioxygenases share significant
amino acid sequence homology, e.g. the two sub-
units which make up the terminal oxidase of the
toluatc dioxygenase and those of the napht ha-
lene, toluene or biphenyl dioxygenase [44,45].
243
Many ot her ar omat i c ring di oxygenases have riot
yet been char act er i zed on t he DNA sequence
level, and compar i son can only be made on t he
basis of bi ochemi cal i nf or mat i on [43].
Anot her class of enzyme activities catalyzing
initial steps in ar omat i c met abol i sm are t he
monooxygenases or hydroxylases. Thes e catalyze
t he i ncor por at i on of a single hydroxyl gr oup on
t he ar omat i c ring or oxidize alkyl side chains.
Several di f f er ent enzyme groups are f ound which
are not significantly r el at ed on ami no acid se-
quence level. Single component ar omat i c ring
f l avopr ot ei n hydroxylases are exempl i fi ed by p-
hydr oxybenzoat e hydroxylase, with a monomer
size of 45 kDa [46]. An overall ami no acid se-
quence i dent i t y of 25% was f ound bet ween p-hy-
dr oxybenzoat e hydroxyl ase and salicylate hydrox-
ylase ( encoded by t he NAH pl asmi d gene nahG)
[47], wher eas 2, 4-di chl orophenol hydroxyl ase (en-
coded by t he gene tfdB of pl asmi d pJP4) [38] and
phenol hydroxyl ase ( encoded by pheA) [48] are
substantially l arger, and only share significant
sequence similarity in two regions, one of which
may be involved in FAD binding. The 2,4-dichlo-
r ophenoxyacet i c acid monooxygenase ( encoded by
tfdA on pl asmi d pJP4) is not r el at ed to t hese
single component monooxygenases [43]. Recent l y
it was r epor t ed, however, t hat t he enzyme is a
ferrous-i on- and c~-ket ogl ut arat e-dependent di-
oxygenase, r at her t han a monooxygenase [49].
Mul t i component ar omat i c ring monooxygenases
are also f ound, e.g. phenol hyxdroxylase f r om
Pseudornonas CF600 ( encoded by t he dmp-
KLMNOP genes) [50], and t ol uene 4-mono-
oxygenase f r om P. mendocina KR1 ( encoded by
t he tmoABCDE genes) [51]. These two enzyme
compl exes have t hr ee pr ot ei n subuni t s in com-
mon [51]. Fur t her mor e, t he TmoC f er r edoxi n and
TodB f er r edoxi n of t he ar omat i c ring dioxyge-
nases shar e 32% ami no acid sequence i dent i t y
and t her ef or e may be of similar evol ut i onary ori-
gin [51]. Anot her class of monooxygenase activi-
ties is f ound in enzymes whi ch oxidize alkyl side
groups on ar omat i c rings, such as xyl ene mono-
oxygenase [52] and t ol uene sul fonat e met hyl mo-
nooxygenase [53]. Thes e two enzymes have bio-
chemi cal l y distinct pr oper t i es, however, and do
not appear strongly r el at ed [53].
Several ot her enzymat i c steps are r equi r ed to
convert ar omat i c subst rat es to t he hydroxyl at ed
i nt er medi at es. Very i nt erest i ng f r om an evolu-
t i onary poi nt of view are t he ext radi ol cl eavage
enzymes, 2, 3-di hydroxybi phenyl 1,2-dioxygenase
( BphC) [54] and 1, 2-di hydroxynapht hal ene dioxy-
genase ( NahC) [33], which share significant over-
all ami no acid sequence similarity with t he cat e-
chol 2, 3-dioxygenases XylE, NahH [33] and DmpB
[55]. Ot her ext radi ol cl eavage enzymes such as
2, 2' , 3-t ri hydroxybi phenyl di oxygenase [56] may
also bel ong to this large pr ot ei n family. Dehydr o-
genases catalyze t he r educt i on of t he di hydrodi ol
compounds f or med by t he activity of t he aromat i c
ring di oxygenases to f or m cat echol s. These dehy-
dr ogenases were shown to be r el at ed to one an-
ot her and to bel ong to t he family of short -chai n
al cohol dehydr ogenases [57]. Uni que enzyme ac-
tivities f ound in t he first steps of ar omat i c
met abol i sm may have been r ecr ui t ed into t hese
pat hways f r om yet unknown evol ut i onary origin,
e.g. dehal ogenat i ng enzymes ( 4- chl or obenzoat e
dehal ogenase of Pseudomonas sp. CBS3 [58] or
dehydr ochl or i nase of P. paucimobilis UT6 [59].
In concl usi on, several classes of enzymes cat-
alyzing t he earl y stages of t r ansf or mat i on of aro-
mat i c compounds are f ound (Fig. 1). Enzymes
within t hese classes, such as t he mul t i - component
dioxygenases, share significant sequence similari-
ties with one anot her . Some of t hem, e.g. t he
ext radi ol di oxygenases appear to form evol ut i on-
ary r el at ed pr ot ei n families with enzymes from
' deeper ' met abol i c branches, such as cat echol
2, 3-dioxygenase. Thi s evol ut i onary r el at edness
and t he genet i c organi zat i on of cat abol i c gene
clusters [6] suggest genet i c processes by which
DNA f r agment s cont ai ni ng several genes (some-
t i mes r ef er r ed to as ' gene modul es' or ' gene
casset t es' ) or gene f r agment s are combi ned to
f or m new met abol i c pat hways or pr ot ei n activi-
ties. An exampl e of t he exi st ence of such gene
modul es may be a DNA f r agment cont ai ni ng t he
genes for t he ar omat i c ring di oxygenase and t he
benzene glycol dehydr ogenase (Fig. 2). Thes e
genes can be f ound at totally di f f er ent posi t i ons
in t he genomes of di f f er ent bact er i a [6]. Ot her
exampl es of some put at i ve gene modul es within
ar omat i c degr adat i on pathways, are t he meta
244
cl eavage pat hway genes (in st ri ct er sense) and t he
genes for a modi f i ed ortho cl eavage pathway.
The meta cl eavage pat hway genes have been
f ound in almost i dent i cal genet i c or gani zat i on as
part of t he oper ons for salicylate degr adat i on
(nah genes), t ol uat e and met at ol uat e degr adat i on
(xyl genes), phenol degr adat i on (dmp genes), and
t ol uene degr adat i on (tod genes) [6]. The reac-
tions cat al yzed by t hese gene modul es are shown
in Fig. 2.
Evolution of the chlorocatechol oxidative pathway
An i mpor t ant cent r al pat hway for t he ar omat i c
degr adat i on of chl or i nat ed compounds in bact e-
ria is t he chl or ocat echol oxidative pat hway or
modi f i ed or t ho cl eavage pat hway [2,41,60]. Chl o-
r i nat ed cat echol s are conver t ed by a specific set
of enzymes to finally 3- oxoadi pat e [61,62], whi ch
may carry one chl ori ne at om, dependi ng on t he
amount of chl ori ne subst i t ucnt s on t he cat echol .
Chl or ocat echol 1,2-dioxygenase, t he first enzyme
of t he pat hway, is an enzyme t hat is approxi-
mat el y 40% i dent i cal to t he nor mal cat echol 1,2-
1.Aromotic ri ng dioxygenose /dehydrogenose
NADI I +H* NAD ~ O H
f~ ~H NAD* ' OH
NADH+W
2. Mo d i f i e d o r t h o cl eavoge pot hwoy
e l (C l ) H C O O H
3. Meto cl eavoge pot hwoy
o
~OH ~ C
C H a I C O O H
> ~oo. > .
O H H
R H O O ~ -.C ~ cH J R
H
Fig. 2. Met abol i c react i ons catalyzed by enzymes encoded on
t hree put at i ve gene modul es. Open arrows bet ween different
i nt ermedi at es indicate t hat more t han one enzymat i c step is
required, closed arrows depict single enzymat i c steps. Black
arrows indicate t he cleavage site for t he catechol dioxygenase.
dioxygenase, but t hat has a much wi der subst rat e
range with respect to conversi on of chl orocat e-
chols [30,63]. Chl or omuconat e cycl oi somerase, t he
next enzyme in t he pathway, can have very di ffer-
ent subst rat e specificities dependi ng on t he strain
from which it was i sol at ed [64]. Chl or omuconat e
cycl oi somerases have a high sequence similarity
t o t he muconat e cycl oi somerase ( appr oxi mat e-
ly 40% overal l ami no acid sequence i dent i t y)
[30]. Some chl or omuconat e cycl oi somerases are
t hought to have an active dechl or i nat i on mecha-
nism, as opposed to t he spont aneous dechl ori na-
tion in t he conversi on of 3- chl or omuconat e by
muconat e cycl oi somerase. The chl orodi enel ac-
t ones which are f or med by t he activity of chl oro-
muconat e cycl oi somerase are t hen f ur t her trans-
f or med by di enel act one hydrol ase and by maley-
l acet at e r educt as e. The l at t er enzyme may also
have a dechl or i nat i ng activity [62].
The genet i c or gani zat i on of t he chl or ocat echol
oxidative pat hway di ffers subst ant i al l y f r om bot h
t hat of t he normal ortho cl eavage pat hway, such
as t hat char act er i zed f r om Acinetobacter cal-
coaceticus, and t hat of t he pr ot ocat echuat e pat h-
way [14,35,36,44]. In t he normal ortho cl eavage
pathway, catA encodi ng cat echol 1,2-dioxygenase,
is separ at ed f r om t he ot her genes of t he pat hway
(Fig. 3A). The onl y genes in t he pr ot ocat echuat e
pat hway with significant similarity t o t hose of t he
modi fi ed ortho pat hway genes, are t he pcaHG
genes encodi ng t he pr ot ocat echuat e 3,4-dioxy-
genase (Fig. 3B). However , t hese show even less
conservat i on in t hei r l ocal i zat i on within t he pat h-
way gene cl ust ers (Fig. 3B). In t he chl or ocat echol
oxidative pat hway, t he gene encodi ng t he chl oro-
cat echol 1, 2-dioxygenase is di rect l y coupl ed to
t he chl or omuconat e cycl oi somerase gene [30]. In-
terestingly, onl y t hese two genes are common to
t he ortho and modi f i ed ortho pathways. Af t er
t he stage of t he cycl oi somerase, t he pathways
diverge. In t he oper ons for t he chl or ocat echol
oxidative pathway, we find evi dence for DNA
r ear r angement s af t er t he chl or omuconat e cyclo-
i somerase gene [30]. Two of t he t hr ee di f f er ent
char act er i zed oper ons have an ext ra DNA frag-
ment bet ween t he genes encodi ng chl or omu-
conat e cycl oi somerase and di enel act one hydro-
lase, wher eas t he ot her has onl y r emnant s of this
245
DNA f r agment . It coul d be t hat t he first par t of
t he oper on, i.e. t he chl or ocat echol 1, 2-di-
oxygenase and chl or omuconat e cycl oi somer ase
genes, became f used wi t h a di f f er ent set of genes
f r om anot her ori gi n, si nce t he l at t er genes have
no appar ent sequence homol ogy wi t h genes f r om
t he nor mal ortho cl eavage pat hway.
A speci al case is r equi r ed for t he di f f er ent
r egul at or y genes whi ch ar e i nvol ved in t he r egul a-
t i on of t he ( chl or o) cat echol oxi dat i ve pat hways.
Regul at or y genes for t he ortho cl eavage pat hways
i ncl ude cat M ( A. calcoaceticus) [65], cat R (P.
put i da) [66,67], t cbR ( Pseudomonas sp. st r ai n P51)
[31], clcR (P. put i da pAC27) [68], and tfdS ( Al -
caligenes eutrophus J MP134) [69,70]. Al l encode
pr ot ei ns whi ch bel ong to t he gr oup of LysR t r an-
scr i pt i onal act i vat or s and ar e l ocat ed at a si mi l ar
posi t i on wi t h r espect t o t he rest of t he pat hway
genes whi ch t hey r egul at e (Fi g. 3), suggest i ng t hat
t hey f or m an anci ent t ype of r egul at i on. Al t hough
Ca t R and CI cR r es pond to t he i nducer muconat e,
it is not known to what ext ent t he var i ous r egul a-
t ors di f f er in t hei r i nducer speci fi ci t y or in r ecog-
ni t i on of DNA- bi ndi ng si t es at t he oper at or . It
will be i nt er est i ng to f ur t her anal yze changes t hat
may have occur r ed in t he di f f er ent modi f i ed
ortho cl eavage pat hways bef or e t hey obt ai ned
t hei r fi nal form and to det er mi ne t he possi bl e
' or i gi nal ' subst r at es for t hi s pat hway in bact er i a.
C o n c l u d i n g r e ma r ks
Compar at i ve st udi es in bact er i a have r eveal ed
evi dence of evol ut i onar y pr ocesses t hat cr eat ed
or modi f i ed di f f er ent met abol i c pat hways, e.g.
A
f
B
o O ; O O ?
COOH
catA M B C
,,ti NL i ,
OH H O O C I ~ co
CI ~ O H -~ CI
OH
E F D
b
I I I
pea E F D B C H G ~
I i I i I
i ~ e "
COOH
B D C E H G
"4 I I ~ 4 - - - = q - - I - 4 - - ~
C
HOOC CI
CI OH
tcbR C D off E F
HOOC CI
OH
CI COOH
c l c R A B orf D
HOOC el
e , ~ o H
C'Lo
Fig. 3. Comparison of the genetic organization of the different ortho cleavage pathways and reactions catalyzed by the intradiol
dioxygenases. (A) Normal ortho cleavage pathway encoded by the catA, catM, and catBCEFD genes of A. calcoaceticus [14,15,65].
(B) Protocatechuate pathway genes of A. calcoaceticus and of P. putida (below) [35,36,94]. (C) Modified ortho cleavage pathways
encoded by the tcbCDEF operon of Pseudomonas sp. strain P51 [29-31l, clcABD of P. putida (pAC27) [40,68], and tfdCDEF of A.
eutrophus JMPI34 [38,39]. The genes encoding the intradiol cleavage enzymes are shown in black; the reactions these enzymes
catalyze are depicted inside the panels. A small arrow indicates a side reaction with less efficiency. Arrows directly above the gene
clusters indicate the direction of transcription. Similar shadings in the genes represent significant sequence similarities between the
genes.
246
t hos e f or de gr a da t i on of xe nobi ot i c c ompounds .
It has be c ome cl ear t ha t s ome s pe c i a l i z e d f unc-
t i ons, e. g. e nz yme s f or c onve r s i on of c hl or i na t e d
a r oma t i c s , pr oba bl y evol ved pr i or t o t he i nt r oduc -
t i on of xe nobi ot i c s i nt o t he e nvi r onme nt . On t he
ot he r ha nd, ne w e vol ut i ona r y e ve nt s s uch as hor i -
z ont a l ge ne t r a ns f e r pr oc e s s e s or poi nt - mut a t i ons
ar e st i l l t a ki ng pl ace a nd c a n ha ve a n i mpor t a nt
i mpa c t on t he a da pt i bi l i t y of s t r ai ns . The que s t i on
of whe t he r t he oc c ur r e nc e of l ar ge qua nt i t i e s of
s ynt het i c, t oxi c c ompounds ha s l ed t o a r a pi d
e vol ut i on of ne w ba c t e r i a l ge not ype s , howe ve r , is
st i l l ope n. Sever al s t udi e s ha ve r e c e nt l y i ndi c a t e d
t ha t mut a t i ons woul d be pos s i bl e i n ba c t e r i a whi c h
ar e ' e nvi r onme nt a l l y i nduc e d' [ 71- 76] , a l t hough
t he i s s ue is s ubj e c t t o de ba t e [ 77, 78] . It will be
ver y i nt e r e s t i ng t o f i nd mor e e vi de nc e of r e gul a -
t or y ci r cui t s i n ba c t e r i a t hat , s e ns i ng c ha ngi ng
e nvi r onme nt s [79] or t he pr e s e nc e of t oxi c com-
pounds [ 80, 81] , s wi t ch on me c ha ni s ms l e a di ng t o
f a vor a bl e ge ne t i c a l t e r a t i ons .
Acknowledgement
1 woul d l i ke t o t ha nk Fl ynn Pi c a r da l f or cr i t i cal
r evi ew of t he ma nus c r i pt .
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