Biodegradation of Aromatic Compounds by Escherichia coli

47 Pages·2001·2.06 MB·English

MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,Dec.2001,p.523–569 Vol.65,No.4
1092-2172/01/$04.00(cid:1)0 DOI:10.1128/MMBR.65.4.523–569.2001
Copyright©2001,AmericanSocietyforMicrobiology.AllRightsReserved.
Biodegradation of Aromatic Compounds by Escherichia coli
EDUARDOD´IAZ,*ABELFERRA´NDEZ,†MAR´IAA.PRIETO,ANDJOSE´ L.GARC´IA
DepartmentofMolecularMicrobiology,CentrodeInvestigacionesBiolo´gicas,ConsejoSuperiordeInvestigaciones
Cient´ıficas,28006Madrid,Spain
INTRODUCTION.......................................................................................................................................................524
TheTwoHabitatsofE.coli...................................................................................................................................524
SourcesofAromaticCompoundsintheE.coliLifeCycle...............................................................................525
IntraspeciesVariationinE.coliandtheCatabolismofAromaticCompounds............................................526
GENERALORGANIZATIONOFTHEGENECLUSTERSFORTHECATABOLISMOFAROMATIC
D
ACIDSANDAMINESINE.COLI..................................................................................................................527
o
4HPA/3HPACatabolicPathway............................................................................................................................527 w
3HPP/3HCICatabolicPathway............................................................................................................................527 n
PPCatabolicPathway............................................................................................................................................529 lo
a
PACatabolicPathway............................................................................................................................................530 d
UpperPathwayfortheCatabolismofAromaticAmines..................................................................................531 e
d
ENZYMESFORTHECATABOLISMOFAROMATICACIDSANDAMINESINE.COLI........................532
f
AromaticRing-HydroxylatingOxygenases..........................................................................................................532 r
o
4HPA/3HPAmonooxygenase.............................................................................................................................532 m
3HPPmonooxygenase.........................................................................................................................................535
h
PPdioxygenase....................................................................................................................................................535 t
t
p
AromaticRingCleavageDioxygenases................................................................................................................535
:
/
HPCmetaCleavageDehydrogenativeRoute.......................................................................................................536 /
m
DHPPmetaCleavageHydrolyticRoute...............................................................................................................538
m
UpperPathwayfortheCatabolismofAromaticAmines..................................................................................539
b
PAAerobicHybridPathway..................................................................................................................................540 r
.
REGULATORYELEMENTSTHATCONTROLEXPRESSIONOFTHEGENECLUSTERSFORTHE a
s
CATABOLISMOFAROMATICACIDSANDAMINESINE.COLI........................................................542 m
TranscriptionalActivators.....................................................................................................................................542 .
o
HpaAprotein.......................................................................................................................................................542 r
g
MaoBprotein.......................................................................................................................................................543 /

MhpRprotein......................................................................................................................................................543 o
n
HcaRprotein.......................................................................................................................................................544
J
TranscriptionalRepressors...................................................................................................................................544 a
n
HpaRprotein.......................................................................................................................................................544
u
PaaXprotein........................................................................................................................................................544 a
r
TRANSPORTPROTEINSOFAROMATICCOMPOUNDSINE.COLI..........................................................545 y

HpaXPermease.......................................................................................................................................................545 8
,
MhpTPermease......................................................................................................................................................546
2
HcaTProtein...........................................................................................................................................................546 0
1
APutativePAPermease........................................................................................................................................546
9
UptakeofOtherAromaticCompounds...............................................................................................................547
b
OTHERENZYMATICACTIVITIESACTINGONAROMATICCOMPOUNDSINE.COLI.......................547 y

CatabolismofHeterocyclicAromaticCompoundsinE.coli............................................................................547 g
u
TryptophanCatabolism.........................................................................................................................................548 e
EnzymaticReactionsinUbiquinoneBiosynthesis.............................................................................................549 s
t
EnzymaticReactionsinEnterobactinandMenaquinoneBiosynthesis..........................................................549
PenicillinGAcylase................................................................................................................................................550
ReductionofNitroaromaticCompounds.............................................................................................................550
ArylamineN-AcetyltransferaseActivity...............................................................................................................552
Arylsulfatase-LikeGenes.......................................................................................................................................552
DehalogenationReactions.....................................................................................................................................552
EVOLUTIONARYCONSIDERATIONSABOUTTHEAROMATICCATABOLICCLUSTERSOF
E.COLI................................................................................................................................................................552
*Correspondingauthor.Mailingaddress:DepartmentofMolecular
Microbiology,CentrodeInvestigacionesBiolo´gicas,ConsejoSuperior
de Investigaciones Cient´ıficas, Vel´azquez 144, 28006 Madrid, Spain.
Phone:[phone].Fax:[phone].E-mail:[email protected].
†Presentaddress:DepartmentofMicrobiology,TheUniversityof
Iowa,IowaCity,IA52242.
523
524 DIAZ ET AL. MICROBIOL.MOL.BIOL.REV.
BIOTECHNOLOGICALAPPLICATIONSOFTHECATABOLISMOFAROMATICCOMPOUNDS
INE.COLI...........................................................................................................................................................556
RelevantPropertiesofE.coliToTackleEnvironmentalPollutionbyAromaticCompounds....................556
Increasedsolventtolerance...............................................................................................................................556
Heavy-metalresistance.......................................................................................................................................557
Aerobic/anaerobiclife-style................................................................................................................................557
Surfacedisplay....................................................................................................................................................557
ExpressionofE.coliAromaticCatabolicGenesinHeterologousHosts........................................................557
hpagenecluster...................................................................................................................................................557
mhpandhcageneclusters.................................................................................................................................558
paagenecluster...................................................................................................................................................558
ExpressionofHeterologousGenesinE.coliforBiodegradationandBiotransformationofAromatic
Compounds..........................................................................................................................................................558
ExpansionoftheabilitiesofE.colitogrowonaromaticcompounds........................................................558
EngineeringofE.colistrainsasbiocatalystsforselectedbiotransformations.........................................560 D
ConstructionofE.coliwhole-cellbiosensorsandcontainedbiocatalysts..................................................561 o
w
CONCLUSIONSANDOUTLOOK..........................................................................................................................561
n
ACKNOWLEDGMENTS...........................................................................................................................................563 lo
REFERENCES............................................................................................................................................................563 a
d
e
d
INTRODUCTION Atfirstview,theabilityofE.colitodegradearomaticcom- f
r
poundsappearstobeanunexpectedfindingsincethisfeature o
m
Nexttoglucosylresidues,thebenzeneringisthemostwidely hasalwaysbeenassociatedwithtypicalsoilbacteriaandE.coli
h
distributedunitofchemicalstructureinnature(59,132).The is mainly regarded as an inhabitant of the animal gut. How- t
t
complexaromaticpolymerlignincomprisesabout25%ofthe ever,whenoneanalyzestheecologyofE.coli,itbecomesclear p
:
land-based biomass on Earth, and the recycling of this and thatthisbacteriummayeasilyencounteraromaticcompounds //m
otherplant-derivedaromaticcompoundsisvitalformaintain- in both the intestinal and extraintestinal habitats that it colo- m
ing the Earth’s carbon cycle. The degradation of such chemi- nizes, which explains its catabolic potential to use such com- b
calsisaccomplishedmainlybymicroorganisms(129,321),and poundsascarbonandenergysource. r.
a
inrecentyearstherehasbeenconsiderableinterestinexplor- s
m
ingtheirabilitytodegradeanddetoxifytheincreasingamounts TheTwoHabitatsofE.coli .
o
of aromatic compounds which enter the environment as by-
r
g
products of many industrial processes (239). Although many Theintestineofwarm-bloodedanimals,theprimaryhabitat
/

generaofmicroorganismsdegradearomaticcompoundsother ofE.coli,containssome400to500differentbacterialspecies, o
n
thanaromaticaminoacids,withPseudomonasbeingthemost withE.colibeingthemostabundant(makingupabout1%of
J
extensively analyzed (132, 335), this ability has been only oc- the total fecal bacterial flora) (233). The success of E. coli in a
n
casionallystudiedinentericbacteria.Theearlyliteraturecon- the gut ecosystem is thought to reflect its abilities to occupy u
tainsreportsontheformationofphenolandp-cresolbyEsch- different ecological niches. Thus, since E. coli grows both a
r
erichia coli bacterial cultures growing in natural media, anaerobically and aerobically, it is able to colonize intestinal y

8
peptone and casein media, and in chemically defined media habitats in which oxygen offers some ecological advantage. ,

containingL-tyrosineorp-hydroxybenzoicacid(316).Thede- Such habitats could be ones in close proximity to epithelial 20
carboxylation of substituted cinnamic acids with the produc- cells, where oxygen molecules might pass from the blood 1
9
tionofvolatilephenoliccompoundsthatprovidephenolicfla- through the epithelium to the microbes attached to it. By
b
vors in fermented beverages has been reported for many assimilating such molecules, E. coli may be important in de- y

enterobacteria of the genera Klebsiella, Enterobacter, and veloping and maintaining the oxygen-free conditions and low g
u
Hafnia,aswellasforEscherichiaintermedia(179).Theuseof oxidation-reduction potential favoring strict anaerobes in the e
benzoic acid, p-hydroxybenzoic acid, and phenylacetic acid largeintestine(280).Whileembeddedwithinthemucuslayer s
t
(PA)byEnterobacteraerogenesasthesolecarbonandenergy overlyingintestinalepithelialcells,E.coligrowswithagener-
source was one of the first reports of the catabolism of aro- ationtimeof40to80min(245,246).Incontrast,thepopula-
matic acids by an enterobacterium (119). In the mid-1970s, tionofE.colicellsinthececalluminalcontentsareessentially
Chapman and coworkers found that most enterics could use static with respect to growth and are excreted in the feces
aromaticcompounds,withtheabilitytoutilizehydroxypheny- (246).
laceticacid(HPA)beingthemostwidespread(38).Itwasthe Although E. coli is a highly successful commensal of the
workofCooperandSkinnerin1980ontheabilityofE.colito intestines of warm-blooded animals as well as a pathogen of
mineralize 3- and 4-hydroxyphenylacetic acids (3HPA and theenteric,urinary,pulmonary,andnervoussystems,thisfac-
4HPA)thatdelineatedforthefirsttimeacompletecatabolic ultative anaerobe must survive and grow outside the animal
pathwayinentericbacteria(50).Later,BurlingameandChap- hosttoeffectsuccessfulinterhostspread.E.coliisnotfamous
man (36) reported that many laboratory strains and clinical forextracorporealexistence,butitnonethelessshareswithits
isolates of E. coli can catabolize various aromatic acids. A soil-inhabitingrelativessuchasKlebsiellaspeciestheabilityto
historical perspective on the mineralization of aromatic com- thrive under a wide range of various physical and chemical
poundsbyE.coliissummarizedinTable1. conditions,includingadaptationforsurvivaloverlongperiods
VOL.65,2001 BIODEGRADATION OF AROMATIC COMPOUNDS BY E. COLI 525
TABLE 1. HistoricalperspectiveonthemineralizationofaromaticcompoundsbyE.coli
Yr Author(s)andlocation Eventa
1980 R.A.CooperandM.A.Skinner(Leicester) FirstreportonapathwayforthecatabolismofaromaticacidsinE.coli
(mineralizationof3HPAand4HPA)
1983 R.P.BurlingameandP.J.Chapman(Minnesota) E.colimineralizesavarietyofaromaticacids(PA,HPA,PP,3HPP,3HCI);
biochemicalcharacterizationofthePPand3HPPcatabolicpathway.
1985 R.A.Cooper,D.C.Jones,andS.Parrot IsolationofthefirstE.colimutantsdefectiveincatabolismofaromatic
(Leicester) acids(PA(cid:2)mutants)
1986 R.P.Burlingame,L.Wyman,andP.J.Chapman IsolationandcharacterizationofE.colimutantsdefectiveinPPand3HPP
(Minnesota) degradation
1987 S.Parrot,S.Jones,andR.A.Cooper(Leicester) FirstreportonthecatabolismofaromaticaminesinE.coli(PEAcanbe
usedasnitrogenandcarbonsource)
1987 N.AbdulrashidandD.P.Clark(Illinois) DegradationoffuransandthiophenesbyE.colimutants
1988 J.R.JenkinsandR.A.Cooper(Leicester) Molecularcloningofthemetacleavageroute(hpcgenes)involvedinHPC
degradation D
1990 D.I.RoperandR.A.Cooper(Leicester) Firstnucleotidesequenceofagene(hpcD)involvedinmineralizationof o
aromaticcompounds(HPA)inE.coli w
1993 D.I.Roper,T.Fawcett,andR.A.Cooper Firstnucleotidesequenceofaregulatorygene(hpcR)thatcontrolsthe n
(Leicester) expressionofanaromaticcatabolicoperon(hpc)inE.coli lo
a
1994 M.A.PrietoandJ.L.Garc´ıa(Madrid) CharacterizationoftheHpaBCmonooxygenasefromE.coli;firstprimary d
structureofaTC-FDM e
1996 M.A.Prieto,E.D´ıaz,andJ.L.Garc´ıa(Madrid) Molecularcharacterizationofacompletearomaticcatabolicpathwayfrom d

E.coli(hpacluster)anditsusetoconstructamobiledegradative fr
o
cassette;expansionofthedegradativeabilitiesofP.putidabyexpressing
m
somehpagenes

1997 M.A.PrietoandJ.L.Garc´ıa(Madrid) Characterizationofthefirstgene(hpaX)responsibleofthetransportofan h
t
aromaticacid(HPA)inE.coli t
p
1997 A.Ferr´andez,J.L.Garc´ıa,andE.D´ıaz(Madrid) Heterologousexpressionofacompletearomaticcatabolicpathway(mhp) :
/
fromE.colitoimprovethecatabolicabilitiesofdifferentenvironmentally /m
relevantbacteria m
1998 E.D´ıaz,A.Ferr´andez,andJ.L.Garc´ıa(Madrid) Molecularcharacterizationofthehcaclusterencodingamulticomponent
b
aromaticinitialdioxygenaseinE.coli r
.
1998 A.Ferr´andez,B.Min˜ambres,B.Garc´ıa,E.R. Molecularcharacterizationofanaerobichybridpathwayforthecatabolism a
Olivera,J.M.Luengo,J.L.Garc´ıa,andE. ofPAinE.coli s
m
D´ıaz(Madrid)
.
o
aAbbreviations:HPA,hydroxyphenylaceticacid;HPC,homoprotocatechuate;3HPP,3-hydroxyphenylpropionicacid;3HCI,3-hydroxycinnamicacid;PA,phenyla- rg
ceticacid;PEA,2-phenylethylamine;PP,phenylpropionicacid;TC-FDM,two-componentnonhemeflavin-diffusiblemonooxygenase. /

o
n

J
of nongrowth (210). Soil, water, sediment, and perhaps food necessaryforittocompetewiththehundredsofotherbacteria a
n
are other habitats of E. coli, and the bacterium might spend with which it shares this habitat, it is likely that aromatic u
comparable times in each of its two main habitats (88, 281). compoundscanalsobeafrequentcarbonsourceforE.coliin ar
y
Whilepollutionfromhumansourcesmaybethemostimpor- the animal gut. Aromatic amino acids and plant constituents
8
tantsourceofE.coliintheenvironment(29,260),thefactthat arethemajorsourcesofaromaticcompoundsinthegastroin- ,

this bacterium was found in pristine tropical waters, where it testinal tract. Minor sources of aromatic compounds in the 20
remained physiologically active and grew at rates dependent human gut include certain steroids and some drugs and food 1
9
onnutrientlevels,suggeststhatitcanbeanaturalinhabitant constituents (additives, colorants, and contaminants) (117). b
intheseenvironmentsandthatitmaybepartofapreviously Estimates suggest that between 3 and 25 g of protein and y

established community (22). E. coli can also replicate and g
peptidesentersthelargeboweleverydayfromthediet,aswell u
surviveinsoilprotozoa.Sinceprotozoaarewidelydistributed as from endogenous sources such as host tissues, bacterial e
insoilsandeffluents,theymayalsoconstituteanenvironmen- s
debris,pancreaticenzymes,andothersecretions(296).Inthe t
tal reservoir for transmission of this enterobacterium (17).
largegut,thesesubstancesaredepolymerizedbyamixtureof
Thus, unlike host-specific or obligate parasites, E. coli is a
residual pancreatic endopeptidases and bacterial proteases
highlyadaptablemicroorganismwithanextensiverepertoireof
and peptidases. The resulting short peptides and amino acids
metabolicandregulatorygenesthatfacilitatethecolonization
then become available for fermentation by many intestinal
ofwidelydifferentenvironments(78).
anaerobes such as Bacteroides, Lactobacillus, Bifidobacterium,
Clostridium,andStreptococcusspecies,generatingawiderange
SourcesofAromaticCompoundsintheE.coliLifeCycle
ofphenolicandindoliccompoundsinaseriesofdeamination,
As stated above, aromatic compounds are highly abundant transamination, decarboxylation and dehydrogenation reac-
in soil and water, and therefore it is obvious that they can tions. Phenol, p-cresol, HPA, hydroxyphenylpropionic acid
constituteanormalcarbonsourceforE.coliwhenthisbacte- (HPP),andhydroxybenzoicacidaretheprincipalproductsof
riumreachesitsextraintestinalhabitat.Althoughitisstillnot tyrosinefermentationinthehumanlargeintestine,whilePA,
knownwhichsubstratesE.coligrowsoninthelargeintestine phenylpropionic acid (PP), and benzoic acid are produced
and which pathways provide it with the metabolic advantage from phenylalanine. PP may, however, also be formed from
526 DIAZ ET AL. MICROBIOL.MOL.BIOL.REV.
tyrosine(296,320).Phenylethylamine(PEA)andtyramineare TABLE 2. AromaticacidsdegradedbydifferentE.colistrainsa
producedfromdecarboxylationofphenyalanineandtyrosine,
Aromaticacidutilizedassolecarbonand
respectively(320).Sincetheproductionofthesearomaticcom- No. energysourceb:
Strain
poundswasinhibitedinthepresenceofareadilyfermentable examined
PA HPA PP 3HPP 3HCI
source of carbohydrate, carbohydrate availability may be a
critical factor affecting aromatic amino acid fermentation in B 1 (cid:2) (cid:1) (cid:1) (cid:1) (cid:1)
C 1 (cid:2) (cid:1) (cid:1) (cid:1) (cid:1)
thelargeintestine(296).Thearomaticcompoundsgenerated K-12 2 (cid:1)c (cid:2) (cid:1) (cid:1) (cid:1)
bytheintestinalanaerobesmeetavarietyoffatesinthebody. W 2 (cid:1) (cid:1) (cid:1) (cid:1) (cid:1)
Thus,theymaybedetoxifiedbyglucuronideorsulfateconju- NCTC5928 1 (cid:1) (cid:2) (cid:1) (cid:1) (cid:1)
gation or may remain unabsorbed and be voided in the feces Clinicalisolates 19 (cid:2) (cid:2) (cid:2) (cid:2) (cid:2)
and urine. They can also completely break down under local 5 (cid:1) (cid:2) (cid:2) (cid:2) (cid:2)
2 (cid:2) (cid:2) (cid:2) (cid:1) (cid:1)
aerobicconditionsinthelargeintestineasaresultoftheaction 8 (cid:2) (cid:2) (cid:1) (cid:1) (cid:1)
ofsomefacultativeanaerobessuchasE.coli,suchthatmono- 1 (cid:2) (cid:1) (cid:2) (cid:2) (cid:2)
and dioxygenases are able to incorporate molecular oxygen 2 (cid:1) (cid:1) (cid:2) (cid:2) (cid:2) D
into the aromatic ring (296, 320). A second major source of 3 (cid:1) (cid:2) (cid:1) (cid:1) (cid:1) ow
aromatic compounds in the animal gut involves the dietary 27 (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) n
lo
plantconstituentssuchasferulicandcaffeicacid,whichresult aModifiedfromreference36. a
in HPP acids (235), as well as different flavonoid glycosides bAbbreviations:HPA,3-and4-hydroxyphenylaceticacid;3HCI,3-hydroxy- d
cinnamicacid;3HPP,3-hydroxyphenylpropionicacid;PA,phenylaceticacid;PP, e
that are ingested in daily quantities of 1 to 2 g byhumans. A phenylpropionicacid. d
collaborativebacterialcatabolism,i.e.,syntrophicinteractions cSomeE.coliK-12derivativessuchasDH5(cid:3),HB101,JM109,CC118,and f
r
DH1donotgrowonPA. o
among microorganisms, of flavonoids in the human gut has
m
been suggested. Thus, a number of obligately anaerobic bac-
h
teriafromthehumanintestinalflora,e.g.,Bacteroidesspecies, t
t
arecapableofcleavingtheglycosidicbondofflavonoids,gen- C (no. 122 of the National Collection of Type Cultures, Lon- p
:
erating the corresponding aglycones such as quercetin, don,UnitedKingdom)isaprototrophF(cid:2)strain(24)andwas //m
kaempferol, naringenin, and catechin (338). These aglycones oneofthefirststrainsshowntobeabletorecombine(actingas m
are then subject to ring cleavage by different bacteria, e.g., a recipient) with K-12 (177). E. coli B is a wild-type E. coli b
r
ClostridiumandEubacteriumspecies,givingrisetoavarietyof strain(67).AmutantofstrainBthatisresistanttoradiations, .
a
aromatic acids, such as 4HPA (from kaempferol), 3,4-dihy- E. coli B/r, has been frequently used in laboratory studies s
m
droxyphenylacetic acid (from quercetin), PA (from naringe- (339).TheW(orWaksman)wild-typestrainofE.coli(ATCC
.
o
nin), and HPP (from tricetin and tricin) (121, 285, 338), that 9637) was apparently isolated from the soil of a graveyard by
r
g
canbeutilizedbyotherintestinalbacteriasuchasE.coli. S.A.Waksman(E.Ron,personalcommunication).Avitamin
/

B auxotroph derivative of the W wild-type strain, E. coli o
12 n
IntraspeciesVariationinE.coliandtheCatabolismof ATCC11105(hereafterreferredasE.coliW)(63),isawell- J
AromaticCompounds knownpenicillinGacylaseproducer.Duringthewritingofthis a
n
review, the 5.4-Mb genome of the enterohemorrhagic E. coli u
PhylogeneticanalyseshaveshownthatE.colistrainsfallinto O157:H7strain(groupD)wasreported(236).Thegenomesof a
r
four main phylogenetic groups (A, B1, B2, and D), where E.coliK-12andO157:H7,twostrainsthatlasthadacommon y

8
virulentextraintestinalstrainsbelongmainlytogroupsB2and ancestorabout4.5millionyearsago,revealedanunexpectedly ,

D,whereasmostcommensalstrainsbelongtogroupA(46).E. complex segmented relationship (236). This diversity within 2
0
coliK-12(groupA)isbyfarthemostextensivelystudiedE.coli theE.colispeciesisreflectedinsignificantdifferencesingene 1
9
strain,anditrepresentsthebest-understoodlivingorganismat contentamongdifferentstrainswhosechromosomesizeranges
b
thebiochemicalandgeneticlevels.Thecompletesequencesof from4.5to5.5Mb(214,262).Genesfoundinmostindividuals, y

the 4.6-Mb genome of two E. coli K-12 derivatives, MG1655 thatis,thecoresetofgenesforthatspecies(coregenepool), g
u
(27; EcoGene database accessible using the Colibri website, arethegenesthatdeterminethosepropertiescharacteristicof e
http://www.genolist.pasteur.fr/Colibri/) and W3110 (202; all members of the species. Additionally, each strain has aux- s
t
GenoBase database accessible at the website http://ecoli.aist iliary genes distributed throughout the genome (flexible gene
-nara.ac.jp/),havebeenreported,andfunctionalgenomicanal- pool),e.g.,pathogenicityislandsandmetabolicpathwaygenes,
ysesarebeingperformed(291).Thewild-typestrainofE.coli whichdeterminepropertiesfoundinsomebutnotallmembers
K-12wasisolatedfromthefecesofaconvalescentdiphtheria ofthespeciesandthatcontributetomaintainingthedynamic
patient in 1922 at Stanford University, and subcultures and genepoolinE.coli(78,125,171,236,262).Inthissense,the
derivativesofthisstrainwerefirstreportedin1944(120).Since E. coli intraspecies variation in the ability to use different
the K-12 strains are unable to colonize the human gut (297), aromatic acids as sole carbon and energy sources (36) (Table
theK-12lineageisconsideredtobetheprototypeofabiolog- 2) is a clear example that E. coli strains possess different ca-
ically safe vehicle for the propagation of many efficient gene- pacitiesforutilizinggrowth-limitingnutrientsandthattheyare
cloningandexpressionsystems.However,thepresenceofK-12 likely to be used to increase the fitness and to expand the
strains among E. coli isolates is extremly low, since no K-12 ecological niches of individual E. coli cells. The work of Bur-
strains were detected among 226 environmental and human lingameandChapman(36)revealedthatallsevenlaboratory
stoolisolatedsamples(29).OtherE.colilaboratorystrainsnot strainstestedgrewusingPP,3HPP,or3-hydroxycinnamicacid
derivedfromK-12are,forinstance,E.coliC,B,andW.E.coli (3HCI)asthesolecarbonandenergysource.However,while
VOL.65,2001 BIODEGRADATION OF AROMATIC COMPOUNDS BY E. COLI 527
E.coliWisalsoabletogrowonPAand4HPAor3HPA,E. ageofHPCbyHPC2,3-dioxygenase(HpaD),togive5-carboxy-
coliK-12andE.coliNCTC5928grewonPAbutnoton4HPA methyl-2-hydroxymuconic semialdehyde (CHMS), which under-
or3HPAandE.coliBandE.coliCgrewon4HPAor3HPA goesdehydrogenationto5-carboxymethyl-2-hydroxymuconicacid
butnotonPA(Table2).Noneofthestrainstestedwasableto (CHM) by the action of the CHMS dehydrogenase (HpaE).
grow on 2HPA, cinnamic acid (CI) or its 2- or 4-hydroxy CHM isomerizes to 5-oxo-pent-3-ene-1,2,5-tricarboxylic acid
derivatives,orwiththe2-or4-hydroxyderivativesofPP(36). (OPET) through a CHM isomerase (HpaF), and the OPET
Among the clinical isolates analyzed, 48 (72%) of 67 could undergoes decarboxylation to 2-hydroxy-hept-2,4-diene-1,7-
grow on at least one of the six aromatic acids tested and 27 dioic acid (HHDD) by the HpaG decarboxylase. The forma-
(40%) could use all six (Table 2). Strains that could grow on tion of 2,4-dihydroxy-hept-2-ene-1,7-dioic acid (HHED)
4HPAcouldalsogrowon3HPA,andthosethatgrewon3HPP through the action of the HpaH hydratase on the product of
alsogrewon3HCI,consistentwithacommoncatabolicpath- theHpaG-catalyzedreactionisfollowedbyitsHpaI-mediated
wayforeachofthesetwopairofcompounds(seebelow). cleavage to give pyruvate and succinic semialdehyde as final
Theaimofthisarticleistoreviewthegeneticsandbiochem- products(Fig.1B).Althoughpyruvateisacentralmetabolite,
D
istryofthecatabolismofaromaticcompoundsinE.coli.Our succinic semialdehyde requires previous dehydrogenation to
o
understandingoftheutilizationofthesecompoundsbyE.coli succinicacidtoentertheKrebscycle,andthisenzymaticstep w
has leapt forward in recent years with the genetic character- isnotencodedwithinthehpacluster(seebelow). n
lo
izationofthecognatecatabolicpathways.Additionalinforma- ThehpacatabolicclusterofE.coliW(Fig.1A)iscomposed
a
tionhasbecomeavailablethroughthesequencingoftheE.coli of 11 genes arranged as follows: (i) eight enzyme-encoding d
e
genome.HomologuesofmanyoftheE.coligenesinvolvedin genes organized in two putative operons, the 4HPA/3HPA d
the catabolism of aromatic compounds can be identified on hydroxylase operon (hpaBC) and the HPC meta-cleavage f
r
o
otherbacterialgenomes,someofwhichhavebeencompletely operon (hpaGEDFHI) homologous to the hpcECBDGH
m
sequencedatthetimeofwriting.Wherepossible,dataderived operonfromE.coliC;(ii)tworegulatorygenes,hpaR(hpcRin
h
fromthesegenomeshavebeenalsoincludedinthisarticleand E. coli C) and hpaA, that control the expression of the meta- t
t
some evolutionary considerations have been pointed out. Fi- cleavageandHPAhydroxylaseoperons,respectively;and(iii) p
:
/
nally,theuseofE.coliasabiocatalystforbiotransformationor the hpaX gene, encoding the 4HPA/3HPA transport protein. /m
biodegradation of aromatic compounds will be addressed. All the genes are transcribed in the same direction with the m
Conclusionsderivedfromthisreviewmayprovideusefulstart- sole exception of hpaR (250) (Fig. 1A). Analysis of the inter- b
r
ingpointsforfutureresearch. genic regions revealed the presence of five REP (repetitive .
a
extragenic palindromic) or PU (palindromic unit) sequences s
m
GENERALORGANIZATIONOFTHEGENECLUSTERS (16). While two REP sequences are located in the largest .
o
FORTHECATABOLISMOFAROMATICACIDS intercistronicregionofthemeta-cleavageoperonbetweenthe r
g
ANDAMINESINE.COLI hpaF and hpaH genes, the other three REP sequences are /
foundatthe3(cid:4)endofsuchoperonandmightactastranscrip- o
AsindicatedintheIntroduction,E.coliisabletomineralize n
tionterminationsignals(Fig.1A).Twoputativehairpinloops
several aromatic compounds (Table 2). In this section, the J
that might act as transcriptional terminators are also located a
generalorganizationofthegeneclustersforthedegradationof n
downstreamofthehpaAgeneandhpaBCoperon(250). u
4HPA, 3HPA, 3HPP, 3HCI, PP, PA, and some aromatic AnalysisoftheregionsflankingthehpaclusterfromE.coli a
aminesisrevised. W showed the presence of several open reading frames (orf ry

8
genes) (250) (Fig. 1A). Downstream of hpaR is located an ,
4HPA/3HPACatabolicPathway incompleteorfthatcorrespondstothetsrgenemappedinthe 2
0
SomeE.colilaboratorystrainssuchasE.coliB,C,andW, chromosomeat98.9min(hereafter,chromosomalmappingin 1
9
but not E. coli K-12, are able to degrade 4HPA, 3HPA, and E.colireferstothatofE.coliK-12strainMG1655[EcoGene
b
homoprotocatechuate(3,4-dihydroxyphenylacetate[HPC])via database at the Colibri website]) and that encodes the serine y

an inducible chromosomally encoded meta-cleavage pathway chemoreceptor(6).Attheotherendofthehpacluster,thatis, g
u
(36,50).ThegenesencodingtheHPCmeta-cleavagedegrada- downstreamofthehpaCgene,thereareseveralorfgenesthat e
tive route from E. coli C (hpc cluster) and E. coli W (hpa correspondtotheyjiY(orf12andorf13)andyjiA(orf14)genes s
t
cluster)havebeenclonedandsequenced(143,250,270).The ofE.coliK-12(EcoGenedatabaseattheColibriwebsite)(Fig.
upper hpa gene cluster encoding the enzymes responsible for 1A).
thehydroxylationof4HPAand3HPAtothecatecholicinter-
mediateHPCinE.coliWhasalsobeenclonedandsequenced, 3HPP/3HCICatabolicPathway
anditislocatedimmediatelyadjacenttotheHPCmeta-cleav-
agegenes(Fig.1A).ThehomologousupperhpaclusterofE. Most E. coli strains are able to degrade 3HPP via a meta-
coliChasalsobeenclonedandpartiallysequenced(251,254). fissionpathway(36)(Table2),andseveralmutantsdefectivein
In addition, near the hpa cluster E. coli W contains the pac thiscatabolismhavebeenisolatedandcharacterized(39).The
gene(Fig.1A),whichencodespenicillinGacylase,anenzyme biochemical pathway for the catabolism of 3HPP in E. coli
able to hydrolyze a wide range of amides and esters of HPA K-12(36,39)isshowninFig.2B.Thefirststepiscatalyzedby
and PA (251, 254) (see below). The biochemical pathway for the MhpA hydroxylase, which inserts one atom of molecular
the catabolism of HPC in E. coli W (250) is similar to that oxygenintothe2positionofthephenylringof3HPPtogive
previouslydelineatedinE.coliC(50,143,270)(Fig.1B).The 2,3-dihydroxyphenylpropionate(DHPP),which,whenactedon
HPC dehydrogenative route in E. coli W involves meta-cleav- by 2,3-dihydroxyphenylpropionate 1,2 dioxygenase (MhpB),
528 DIAZ ET AL. MICROBIOL.MOL.BIOL.REV.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
m
b
r
.
a
FIG. 1. PathwayforthecatabolismofHPA(4HPAand3HPA)inE.coli.(A)Geneticmapofthechromosomalhpa(inE.coliW)andhpc s
(inE.coliC)regions.Relevantgenesareindicatedbyblocks:geneswithsimilarshadingparticipateinthesameenzymaticsteporinthesame m
functionalunit(route)ofthepathway.Thehpcgenesareindicatedinbrackets.Regulatoryandtransportgenesareshownbysolidandvertically .
o
stripedblocks,respectively.Thegenesflankingthehpacluster(tsr,orf12,orf13,andorf14)arecontiguousinthegenomeofE.coliK-12andare r
g
representedbythicklines.orf12andorf13correspondtotheyjiYgenefromE.coliK-12.orf14correspondstotheyjiAgenefromE.coliK-12.The
/
arrowsshowthedirectionsofgenetranscription.BentarrowsrepresentthePr,Pg,Px,Pa1,Pa2andPBCpromoters.REPsequencesareshown. o
TheHpaRrepressorandHpaAactivatorarerepresentedbyasquareandhexagon,respectively;emptyandsolidsymbolsindicateinactiveand n
activeregulators,respectively;(cid:2)and(cid:1)indicatetranscriptionalrepressionandactivation,respectively.Theinducermolecule(HPAandHPC)is J
a
representedbyasolidcircle.(B)BiochemistryoftheHPAcatabolicpathway.Themetabolitesare4HPAand3HPA,HPC(homoprotocatechuate),
n
CHMS (5-carboxymethyl-2-hydroxy-muconic semialdehyde), CHM (5-carboxymethyl-2-hydroxy-muconic acid), OPET (5-oxo-pent-3-ene-1,2,5- u
tricarboxylicacid),HHDD(2-hydroxy-hept-2,4-diene-1,7-dioicacid),OHED(2-oxo-hept-3-ene-1,7-dioicacid),andHHED(2,4-dihydroxy-hept- a
r
2-ene-1,7-dioic acid). The enzymes are HpaBC (HPA monooxygenase), HpaD (HPC 2,3-dioxygenase), HpaE (CHMS dehydrogenase), HpaF y
(CHMisomerase),HpaG(OPETdecarboxylase),HpaH(hydratase),HpaI(HHEDaldolase),andSad(succinicsemialdehydedehydrogenase). 8
TheHPAtransportprotein(HpaX)isrepresentedbyathickarrow.OutandInindicateoutsideandinsidethecell,respectively. ,
2
0
1
9
undergoesanextradiolcleavage,yieldingthemetaringfission sole exception of mhpR (Fig. 2A). Although the Shine-Dal-
b
product, 2-hydroxy-6-keto-nona-2,4-diene 1,9 dioic acid garno sequences of the mhpR, mhpA, and mhpC genes are y

(HKNDA).HKNDAiscleavedbytheMhpChydrolasetogive locatedinintergenicregions,thoseofmhpB,mhpD,mhpF,and g
u
succinate and 2-hydroxy-penta-2,4-dienoic acid (HPDA), mhpE overlap the preceding genes, suggesting that the most e
whichishydratedto4-hydroxy-2-ketopentanoicacid(HKP)by commonmechanismoftranslationalcouplingmayoccur(92). s
t
the MhpD hydratase. Then the MhpE aldolase catalyzes the The mhp cluster maps at min 8.0 of the chromosome, be-
fission of HKP to give pyruvate and acetaldehyde, with the tweenthelacIgene,encodingthetranscriptionalrepressorof
latter being converted to acetyl coenzyme A (acetyl-CoA) thelacoperon,andtheadhCgene,encodinganalcohol-acet-
through the action of the MhpF acetaldehyde dehydrogenase aldehyde dehydrogenase, that is in the vecinity of the tau
(Fig.2B). operonfortaurinemetabolism(EcoGenedatabaseattheCo-
Analysisofthe9.8-kbmhpcluster(92)(Fig.2A)revealsthe libri website). The order of the catabolic genes in the meta-
existence of eight genes arranged as follows: (i) six genes en- cleavage operon, with the single exception of mhpF, parallels
coding the initial monocomponent monooxygenase (mhpA), thatoftheenzymaticstepsinthe3HPPcatabolicpathway(92)
the extradiol dioxygenase (mhpB), and the hydrolytic meta- (Fig.2).
cleavage enzymes (mhpCDFE); (ii) one regulatory gene ItisknownthatE.coliisalsoabletogrowwith3HCIasthe
(mhpR);and(iii)onegene(mhpT)thatencodesatransporter sole carbon and energy source (36) (Table 2). Growth with
and is flanked by two bacterial interspersed mosaic elements 3HCIinducesthesynthesisoftheMhpAandMhpBenzymes,
(BIMEs) that belong to the BIME-2 subfamily (16). All the whichareresponsiblefortheinitialattackon3HPP(36),and
genesappeartobetranscribedinthesamedirection,withthe ithasbeenshownthatthemhpclusterfromE.coliconfersto
VOL.65,2001 BIODEGRADATION OF AROMATIC COMPOUNDS BY E. COLI 529
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
m
b
r
.
a
s
m
.
o
r
g
FIG. 2. Pathwayforthecatabolismof3HPPinE.coli.(A)Geneticmapofthechromosomalmhpcluster.Relevantgenesareindicatedby /
o
blocks:geneswithsimilarshadingparticipateinthesameenzymaticsteporinthesamefunctionalunit(route)ofthepathway.Regulatoryand n
transportgenesareshownbysolidandverticallystripedblocks,respectively.Genesflankingthemhpcluster(lacIandyaiL)arerepresentedby
J
thicklines.Thearrowsshowthedirectionsofgenetranscription.BentarrowsrepresentthePrandPapromoters.ThelocationoftheBIMEis a
shown.TheinactiveandactiveformsoftheMhpRactivatorarerepresentedbyemptyandsolidhexagons,respectively.(cid:1)indicatestranscriptional n
u
activation. The inducer molecule (3HPP and 3HCI) is represented by a solid circle. (B) Biochemistry of the 3HPP catabolic pathway. The a
metabolitesare3HPP,DHPP(2,3-dihydroxyphenlypropionicacid),HKNDA(2-hydroxy-6-keto-nona-2,4-diene1,9-dioicacid),HPDA(2-hydroxy- r
y
penta-2,4-dienoic acid), and HKP (4-hydroxy-2-ketopentanoic acid). The enzymes are MhpA (3HPP monooxygenase), MhpB, (DHPP 1,2
8
dioxygenase),MhpC(HKNDAhydrolase),MhpD(HPDAhydratase),MhpE(HKPaldolase),andMhpF(acetaldehydedehydrogenase[acylat- ,

ing]).The3HPPtransportprotein(MhpT)isrepresentedbyathickarrow.OutandInindicateoutsideandinsidethecell,respectively. 2
0
1
9

SalmonellaentericaserovarTyphimuriumLT2,astrainunable fragment that carries the hca cluster for PP catabolism re- b
y
to grow on 3HPP and 3HCI, the ability to use these two vealed(i)fivegenesencodingthePP-dioxygenase(hcaEFCD;
g
aromatics as the sole carbon and energy source (92). Hence, formerlynamedashcaA1A2CD)andPP-dihydrodioldehydrog- u
e
themhpgenesarealsoresponsibleforthecatabolismof3HCI enase (hcaB), (ii) a regulatory gene (hcaR), and (iii) a gene s
via2,3-dihydroxycinnamicacid(DHCI)withtheformationof (hcaT) that might encode a transporter (71). Downstream of t
pyruvate,acetyl-CoA,andfumarate(Fig.3). hcaD,thecloselylinkedorfX(Fig.4A)couldalsobelongtothe
hcacluster,anditcodesfora155-amino-acid(aa)productof
PPCatabolicPathway unknown function. The Shine-Dalgarno sequences of hcaE,
The catabolism of PP in E. coli is initiated by a dioxygeno- hcaF, hcaC, hcaB, and hcaD overlap the preceding genes,
lyticpathway(36,39)(Fig.4B).Thefirststepiscatalyzedbya suggestingthattranslationalcouplingoccurs(71).Immediately
PP dioxygenase, which inserts an atoms of molecular oxygen downstreamoforfXthereisaninvertedrepeatsequencethat
intoeachofpositions2and3ofthephenylringofPP,yielding couldactasatranscriptionalterminatorofapotentialoperon.
cis-3-(3-carboxyethyl)-3,5-cyclohexadiene-1,2-diol (PP-dihydro- hcaR and hcaT are located upstream of hcaEFCBDorfX, but
diol), which is subsequently oxidized by the PP-dihydrodiol they are transcribed in the opposite direction from the other
dehydrogenasetogiveDHPP(36,39)(Fig.4B).DHPPisthe genes(Fig.4A).AlthoughtheintergenicspacingbetweenhcaR
substrate of the meta-cleavage pathway described above for andhcaTwas159bp,notypicaltranscriptionalterminatorand
3HPPdegradation,andthereforeitlinksthecatabolismofPP promotersequencesweredetectedinthisDNAfragment(71).
and3HPPinE.coli.Thenucleotidesequenceofa7.2-kbDNA The hca cluster maps immediately downstream of csiE,
530 DIAZ ET AL. MICROBIOL.MOL.BIOL.REV.
D
o
w
n
lo
a
d
e
d

f
r
FIG. 3. Biochemistryofthe3HCIcatabolicpathway.Themetabolitesare3HCI,DHCI(2,3-dihydroxycinnamicacid),HKNTA(2-hydroxy-6- o
keto-nona-2,4,7-triene 1,9-dioic acid), HPDA (2-hydroxy-penta-2,4-dienoic acid) and HKP (4-hydroxy-2-ketopentanoic acid). The enzymes are m
MhpA(3HCImonooxygenase),MhpB,(DHCI1,2-dioxygenase),MhpC(HKNTAhydrolase),MhpD(HPDAhydratase),MhpE(HKPaldolase), h
andMhpF(acetaldehydedehydrogenase[acylating]).The3HCItransportprotein(MhpT)isrepresentedbyathickarrow.OutandInindicate t
t
outsideandinsidethecell,respectively. p
:
/
/
m
m
whichencodesthestationary-phase-inducibleproteinCsiE,at follow the conventional routes for the aerobic catabolism of b
r
57.5minoftheE.colichromosome(EcoGenedatabaseatthe aromaticcompounds(suchasthoseofE.coliforHPA,3HPP, .
a
Colibriwebsite)andthereforefarfrommin8,wherethemhp and PP degradation) and whose first step is the activation of s
m
cluster responsible for DHPP degradation is located (see PAtophenylacetyl-coenzymeA(PA-CoA)bytheactionofa
.
o
above). PA-CoAligase(94,328,182)(Fig.5B).Thesecondstepofthe
r
g
TheHcaEFCDdioxygenaseandHcaBdihydrodioldehydro- PA catabolic pathway will probably involve the hydroxylation
/

genaseareabletotransformCIintoDHCI(Fig.4B)(36,71). of PA-CoA followed by cleavage of the aromatic ring and o
n
Thus,whenthehcaandmhpclustersfromE.coliareexpressed further degradation of the resulting aliphatic compound
J
in S. enterica serovar Typhimurium LT2, the resulting strain through a (cid:5)-oxidation-like pathway (Fig. 5B). Since the par- a
n
cangrowonminimalmediumcontainingCIasthesolecarbon ticipationofCoAligasesintheinitialstepofthecatabolismof u
andenergysource(71).Therefore,whileinsomesoilPseudo- aromatic compounds is a typical feature of anaerobic catabo- a
r
monasspeciesandinLactobacilluspastorianusthecatabolism lism (131), the aerobic PA degradation in E. coli constitutes y

8
of CI could be accomplished by an initial reduction of the oneofthefewexamplesofaerobichybridpathways(94,182). ,

double bond of the side chain with formation of PP, the ca- The paa genes responsible for PA degradation in E. coli 2
0
tabolism of CI by the Hca enzymes in serovar Typhimurium K-12 and E. coli W strains are organized as a chromosomal 1
9
produces DHCI, which, following the mhp-encoded pathway, cluster of about 14 kb (Fig. 5A), which is absent in the PA-
b
willbefinallymineralizedtopyruvate,acetyl-CoA,andfuma- deficientE.coliCstrain.However,theabilityofE.coliK-12to y

rate(71)(Fig.3).SinceCIcaninducetheexpressionofthehca growonPAisstraindependent,withpointmutationsorsmall g
u
genesandisconvertedtoDHCIbyE.colicellsinaresting-cell generearrangementsinthepaaclusterbeingthemostproba- e
process, it is difficult to explain the unexpected observation ble reason for the PA-deficient phenotype of some K-12 lab- s
t
thatthisbacteriumdoesnotgrowwhencultivatedonCIasthe oratorystrains(94)(Table2).
solecarbonandenergysource(71).Apossibleexplanationof TheE.colipaaclustercontains14genesorganizedinthree
this lack of growth could be that the DHCI generated in the transcriptional units; two of them, paaZ and paaABCDEFG
reactions catalyzed by the Hca enzymes (and not the DHCI HIJK, encode the catabolic genes, and the third, paaXY, con-
generated from 3HCI by the MhpA hydroxylase) or other tainsthepaaXregulatorygene.Allthegenesaretranscribedin
intermediates farther down the mhp-encoded pathway accu- thesamedirectionwiththesoleexceptionofpaaZ(Fig.5A).
mulatetoatoxiclevelthatpreventsthenormalmetabolicflux LocateddownstreamofpaaZ,paaK,andpaaY,threeinverted-
of the cell (71). Why this toxicity is not observed in serovar repeat sequences may act as transcriptional terminators (94).
Typhimuriumisalsoanopenquestion. AlthoughthepaaKgeneproductcatalyzesthefirstenzymatic
stepofthePAcatabolicpathway,paaKislocatedatthe3(cid:4)end
PACatabolicPathway ofthepaaoperon(Fig.5A),agenearrangementthatexplains
whyTn1000insertionswithinthepaacatabolicoperoncaused
E.coliWandK-12,butnotE.coliBandC,mineralizePA polareffectsleadingtoE.colimutantsthatdidnotattackPA
(Table 2) through a novel catabolic pathway which does not (94). The paa genes map at the right end of the mao cluster
VOL.65,2001 BIODEGRADATION OF AROMATIC COMPOUNDS BY E. COLI 531
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
FIG. 4. PathwayforthecatabolismofPPinE.coli.(A)Geneticmapofthechromosomalhcacluster.Relevantgenesareindicatedbyblocks: m
geneswithsimilarshadingencodethesubunitsofthePPdioxygenase.Regulatory(solidblock)andputativetransport(verticallystripedblock)
b
genesarealsoshown.ThehorizontallystripedblockindicatesthegeneencodingthePPdihydrodioldehydrogenase.Theemptyblockrepresents r
.
a gene of unknown function. The csiE gene flanking the hca cluster is represented by a thick line. The arrows show the directions of gene a
transcription.BentarrowsrepresentthePrandPepromoters.TheinactiveandactiveformsoftheHcaRactivatorarerepresentedbyemptyand s
solid hexagons, respectively. (cid:1) indicates transcriptional activation. The inducer molecule (PP and CI) is represented by a solid circle. (B) m
.
BiochemistryofthePPcatabolicpathway.ThemetabolitesarePP,CI,PPdihydrodiol,CI-dihydrodiol,DHPP,andDHCI(seethelegendstoFig. o
r
2and3).TheenzymesareHcaEFCD(PPdioxygenase),HcaB(PP-dihydrodioldehydrogenase),andMhpBCDEF(seethelegendtoFig.2).The g
putativePP/CItransportprotein(HcaT)isrepresentedbyathickarrow.OutandInindicateoutsideandinsidethecell,respectively. /
o
n

J
(Fig.5A),whichisinvolvedinthetransformationofPEAinto (PAL) and a phenylacetaldehyde dehydrogenase (PadA or a
n
PA (see below), at 31.1 min near the replication terminus of FeaB) that oxidizes the latter to PA (228) (Fig. 6B). E. coli u
a
the chromosome (94). This location confirms previous obser- mutants defective in PadA cannot grow on PEA as a carbon r
y
vations that mapped the mutations in two PA-deficient mu- sourcebutcanstilluseitasanitrogensource(228).Tyramine
8
tantsofE.coliK-12inthischromosomalregion(48).Although anddopaminearealsosubstratesofMaoAandPadA,leading ,
2
the left end of the paa cluster is adjacent to the maoA gene to formation of the corresponding aromatic acids, i.e., 4HPA 0
bothinE.coliWandK-12,therightendofthisclusterdiffers andHPC,respectively(228)(Fig.6B).Therefore,whileE.coli 19
in these two strains. Thus, while the paaY stop codon was K-12, which lacks the hpa cluster (see above), is able to use b
found 231 bp upstream of the ATG start codon of the ydbC tyramine and dopamine only as nitrogen sources, E. coli W, y
gene in E. coli W (94), a 9.2-kb sequence containing a long g
whichcatabolizes4HPAandHPC,canusethesetwoaromatic u
open reading frame (ydbA) disrupted by two insertion se- e
amines as both carbon and nitrogen sources. The reaction
s
quences(IS2DandIS30C)wasfoundbetweenpaaYandydbC catalyzedbyMaoAleadstotheformationofH O ;therefore, t
inE.coliK-12(EcoGenedatabaseattheColibriwebsite)(Fig. 2 2
toavoidthetoxiceffectsofthelatter,catalaseisalsoinduced
5A).Thepresenceofinsertionsequencesnearthepaacluster
withinE.colicellsgrowinginthepresenceofaromaticamines
andthelocationofthisclusterinanonessentialregionofthe
(228).
chromosome(135)providesomecluestothepossiblemecha-
ExpressionofthemaoAandpadAgenesiscontrolledbythe
nisms of gene mobilization of a catabolic cassette and could
MaoB (FeaR) regulator. The maoA, padA, and maoB genes
explain the heterogeneity observed among different E. coli
(alternative gene names, tynA, feaB, and feaR, respectively)
strainstomineralizePA(seebelow).
have been sequenced in E. coli K-12 and W strains and are
located at 31.1 min on the chromosome (94, 127, 307). The
UpperPathwayfortheCatabolismofAromaticAmines
padA gene is transcribed in the opposite direction to that of
Growth of E. coli on PEA as the sole carbon and energy maoA and maoB (Fig. 6A), indicating that these three genes,
sourceinducestwoenzymaticactivitiesthatconstitutetheup- although involved in the same metabolic pathway and physi-
perpathwayforthecatabolismofaromaticamines:anamine callyassociated,donotconstituteanoperon(95).Sincethese
oxidase (MaoA) that converts PEA into phenylacetaldehyde genes map immediately adjacent to the left end of the paa
532 DIAZ ET AL. MICROBIOL.MOL.BIOL.REV.
D
o
w
n
lo
a
d
e
d

f
r
o
m

FIG. 5. PathwayforthecatabolismofPAinE.coli.(A)Geneticmapofthechromosomalpaacluster.Relevantgenesareindicatedbyblocks: h
geneswithsimilarshadingparticipateinthesameenzymaticsteporinthesamefunctionalunitofthepathway.Genesflankingthepaacluster tt
p
(maoAandydbC)arerepresentedbythicklines.Thearrowsshowthedirectionsofgenetranscription.BentarrowsrepresentthePz,Pa,andPx :
/
promoters.ThelocationsoftheIS2andIS30insertionsequenceswithinydbAinE.coliK-12areshown.Theregulatorygene(paaX)isrepresented /
m
byablackblock.TheinactiveandactiveformsofthePaaXrepressorareindicatedbyemptyandsolidsquares,respectively.(cid:2)and(cid:1)indicate
m
transcriptionalrepressionandactivation,respectively.Theinducermolecule(PA-CoA)isrepresentedbyasolidcircle.(B)Biochemistryofthe
b
PAcatabolicpathway.ThefirstintermediateofthepathwayisPA-CoA(phenylacetylCoA).TheenzymesarePaaK(PA-CoAligase),PaaABCDE r
(putativemulticomponentoxygenase),PaaZ(putativering-cleavageenzyme),andPaaFGHIJ(putative(cid:5)-oxidation-likeenzymaticsystem). .a
s
m
.
o
cluster(Figs.5Aand6A),thissupraoperonicclusteringoftwo component monooxygenase that initiates the catabolism of
r
g
relatedpathways,i.e.,theupperpathwayforPEAcatabolism 4HPAand3HPAinthisstrain(251)(Fig.1).Thisenzymeisa
/

andthePAcatabolicpathway,representsanexampleofphys- member of the two-component nonheme flavin diffusible o
n
icallinkagewithinthePA-CoAcatabolon(182,217). monooxygenases (TC-FDM) family, which can be defined ac-
J
cording to the following properties: (i) the reductase and the a
n
ENZYMESFORTHECATABOLISMOFAROMATIC oxygenasecomponentsareencodedbytwodifferentgenes;(ii) u
ACIDSANDAMINESINE.COLI the reductase component uses NAD(P)H to catalyze the re- ar
ductionofaflavinthatdiffusestotheoxygenasecomponentfor y

The success of a particular catabolic pathway depends on 8
oxidation of the substrate by molecular oxygen; and (iii) the ,
twomajorelements,i.e.,thecatabolicenzymes,whichmustbe two components are not flavoproteins and lack typical ferre- 2
assembled in an optimal chain of sequential transformations 0
doxinand/orflavin/NAD(P)Hbindingmotifs(104). 1
leadingtothemineralizationofthecompound,andtheregu- 9
The HpaBC monooxygenase is the best-studied ring-hy-
latory elements. In this section we review the different cata- b
droxylating oxygenase in the catabolism of aromatic com- y
bolicgenesandthecorrespondingencodedproteinsthatallow
pounds in E. coli. E. coli K-12 strains expressing the hpaBC g
theactivationandcleavageofthearomaticring,aswellasthe u
genesfromaplasmidproducedblackpigmentswhentheywere e
finutrothtehredKegrreabdsactyiocnleo.fAtlhtheonuognhartohmeacatitcaibnotleircmgeednieasteosfftohreeanrtory- growinginminimalglucosemediumsupplementedwithL-Tyr, st
N-acetyl-L-Tyr, L-Tyr-methyl ester, 4HPA, 3HPA, or phenol.
maticbiodegradativepathwaysinE.colihavebeencharacter-
Sincecatecholderivativesformspontaneouslyblackorbrown
ized, most of the catabolic steps for PA degradation still re-
oxidation products, the appearance of a black pigment in the
quireanexperimentaldemonstration.
culture medium reflected the HpaBC-mediated monooxygen-
ationofthearomaticcompoundsaddedtothegrowthmedium
AromaticRing-HydroxylatingOxygenases
(254).ThesubstraterangeoftheHpaBCmonooxygenasewas
The initial step in the aerobic catabolism of 4HPA/3HPA, checked by measuring NADH oxidation. Although the best
3HPP/3HCI,andPPinE.coliiscarriedoutbyoxygenasesthat substratewas4HPAfollowedby3HPA,theenzymewascapa-
introduceone(monooxygenases)ortwo(dioxygenases)atoms ble to oxidize NADH in the presence of chloro- and methyl-
of molecular oxygen into the phenyl ring. These oxygenases phenolssuchas3-chloro-4HPA,4-chloro-PA,4-chlorophenol,
belongtothreedifferentfamiliesofring-hydroxylatingoxyge- 3-chlorophenol,andp-cresol.TheHpaBCmonooxygenasecan
nases(Table3). alsooxidizesomedihydroxylatedaromaticcompoundssuchas
4HPA/3HPA monooxygenase. The hpaBC genes located at HPC, 2,5-dihydroxyphenylacetic acid, and, with lower effi-
the 3(cid:4) end of the hpa cluster in E. coli W encode the two- ciency,catechol,resorcinol,hydroquinone,and3,4-dihydroxy-