5b in situ biodeg and omics

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Metabolic networks, microbial ecology and omics

technologies: towards understanding in situbiodegradation processesemi_2340 1..16

Ramiro Vilchez-Vargas,1 Howard Junca2 and

Dietmar H. Pieper1*1Microbial Interactions and Processes Research Group,

HZI Helmholtz Centre for Infection Research,

Inhoffenstrae 7, D-38124 Braunschweig, Germany.2GeBiX Colombian Center for Genomics and

Bioinformatics of Extreme Environment and Research

Group Microbial Ecology: Metabolism, Genomics and

Evolution of Communities of Environmental

Microorganisms, CorpoGen, Carrera 5 # 66A-35,

Bogot, Colombia.

Summary

Microbial degradation is the main mechanism respon-

sible for the recovery of contaminated sites, where a

huge body of investigations is available in which

most concentrate on single isolates from soilscapable of mineralizing pollutants. The rapid develop-

ment of molecular techniques in recent years allows

immense insights into the processes in situ, includ-

ing identification of organisms active in target sites,

community member interactions and catabolic gene

structures. Only a detailed understanding of the func-

tioning and interactions within microbial communi-

ties will allow their rational manipulation for the

purpose of optimizing bioremediation efforts. We will

present the status of the current capabilities to

assess and predict catabolic potential of environmen-

tal sites by applying gene fingerprinting, catabolome

arrays, metagenomics and complementary omics

technologies. Collectively, this will allow tracking

regulation and evolution within microbial communi-

ties ultimately aiming to understand the mechanisms

taking place in large scale bioremediation treatments

for aromatic decontamination.

Introduction

Given the widespread contamination with aromatic and

aliphatic pollutants, it is a long-held desire to treat

organic and inorganic waste more efficiently and

remediate polluted environments via controllable andamenable microbial activities. However, despite their

promising performance in the laboratory, the application

of pollutant-degrading bacteria in microcosms or near-

field situations have mostly ended in disappointment (El

Fantroussi and Agathos, 2005; Thompson et al., 2005).

Therefore, more optimal and rational use of the

extremely high potential of catalytic activities in the envi-

ronment has been proposed for more successful pollu-

tion treatment (Watanabe et al., 2002). Presently, this

potential cannot be sufficiently exploited because of the

lack of knowledge on the desired catabolic activity and

ecological behaviour of the microbial community (Paerl

and Steppe, 2003). Pollutant degradation in contami-

nated environments is in many cases carried out by

microbial food webs rather than single species (de

Lorenzo, 2008), where key species and catabolic genes

are often not identical to those that have been isolated

and described in the laboratory (Jeon et al., 2003; Witzig

et al., 2006). We now know that microbial diversity in

these environments is in orders of magnitude higher

than assumed from previous cultivation efforts (Leigh

et al., 2007). A particularly large number of novel tech-

niques have been developed, which now allow the

determination of microbial diversity and activity in situ at

the polluted site, straightforward screenings for particulargene diversity, gene quantification, whole-genome

sequencing of bacterial isolates and of DNA and mRNA

from total communities. More knowledge on the potential

of indigenous microbial metabolism of pollutants, on the

processes involved and on the diversity and ecology of

the organisms would permit us to more precisely under-

stand the long-term fate of pollutants and to better direct

our efforts to sustainable decontamination/detoxification

of polluted environments.

Received 2 May, 2010; accepted 5 August, 2010. *For correspon-dence. E-mail [email protected]; Tel. (+49) 531 6181 4200; Fax(+49) 531 6181 4499.

Environmental Microbiology (2010) doi:10.1111/j.1462-2920.2010.02340.x

2010 Society for Applied Microbiology and Blackwell Publishing Ltd
mailto:[email protected]:[email protected]


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Fig.

1.

Aerobicmetabolism

ofaro

maticsviadi-ortrihydroxylatedintermediate

s,

orviaCoAderivatives

.Peripheralhydroxy

lationreactionscanbecatalysedbyflavoproteinmonooxygenases

(FPM),Rieskenon-haem

ironoxy

genases(RNHO

,rearomatizationreactionscatalysedbydihydrodioldehydrogenasesare

notindicated)orsolublediironmonooxygenases(SDM).

Alternatively

,aromaticscanbeac

tivatedthroughCoAligasesfollowedbydearomatizationcatalysedbymembersoftheFP

M

orSDM

.Centraldi-ortrihydroxylatedintermediatesaresubject

toringcleavagebyintradioldioxygenases(INDO)orextradioldioxygenaseso

fthevicinalchelatesuperfamily(EXDO),the

LigBsuperfamily(LigB)orthecupinsuperfamily(CUP).

Ring-c

leavageproductsarechann

elledtotheKrebscycleviacentralreactions(hollow

arrows).

Understanding in situ biodegradation 3

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology


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encoded catechol 2,3-dioxygenase. However, toluene can

also be disassembled via successive monooxygenations

catalysed by soluble diiron monooxygenases with meth-

ylphenols and methylcatechols as intermediates (Leahy

et al., 2003) or through the action of a Rieske non-haemiron oxygenase of the toluene/isopropylbenzene/

biphenyl subfamily followed by dehydrogenation with

toluene dihydrodiol and 3-methylcatechol as intermediates

(Gibson and Parales, 2000). Genes encoding these previ-

ously mentioned enzymes of archetype strains are typi-

cally clustered with genes encoding a broad substrate

specificity extradiol dioxygenase of subfamily I.3.B (Beil

et al., 1999) or an extradiol dioxygenase of subfamily I.3.A

(Eltis et al., 1992) (Fig. 2). Thus, it may seem that analys-

ing the abundance and diversity of respective genes is

appropriate forcharacterizing the potential of a given soil to

degrade toluene and related compounds such as benzene

via a dioxygenolytic route. However, the respective gene

clusters typically comprise only one of the two knownbranches of the meta-cleavage pathway for further disas-

sembling of the ring-cleavage product. The so-called

hydrolytic branch encoded by the respective clusters is

necessary for the degradation of substituted catechols

such as 3-methylcatechol or 2,3-dihydroxybiphenyl where

the ring-cleavage product is a ketone, which is hydrolysed

to 2-hydroxopenta-2,4-dienoate and acetate, in the case

of 3-methylcatechol degradation. Benzene degradation,

in contrast, necessitates the oxalocrotonate branch

Fig. 2. Dendrogram showing the relatedness of extradiol dioxygenases of the vicinal chelate superfamily. Subfamily designations as defined

by Eltis and Bolin (1996) are given.

4 R. Vilchez-Vargas, H. Junca and D. H. Pieper



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(Harayama et al., 1987) whereby intermediate 2-

hydroxymuconic semialdehyde (generated from catechol)

is subject to oxidation by 2-hydroxymuconic semialdehyde

dehydrogenase. In accordance, it has previously been

reported that benzene degrading isolates from a contami-

nated site recruit a pathway comprising a subfamily I.2.A

extradiol dioxygenase which is typically clustered with

such a branch and that subfamily I.2.A extradiol dioxyge-

nases are predominant at the respective site (Witzig

et al., 2006). Surveys that characterize the catabolic

potential for biodegradation thus have to take into consid-

eration the broad diversity of catabolic routes evolved by

microorganisms.

However, this not only holds for the diversity of path-

ways that can be recruited, but also for the diversity of

enzymes of a given gene family or even between gene

families. Even though most biphenyl degrading Actino-

bacteria and Proteobacteria employ an enzyme of the

subfamily I.3.A or I.3.B, the ring cleavage of 2,3-

dihydroxybiphenyl may be catalysed by quite distinctenzymes belonging to different branches of the vicinal

chelate superfamily (Taguchi et al., 2004), which may

even be crucial for degradation (Hatta et al., 2003). Also,

the only distantly related so-called one-domain extradiol

dioxygenases such as BphC2 and BphC3 from Rhodo-

coccus globerulus P6 have reported activity against 2,3-

dihydroxybiphenyl (Asturias and Timmis, 1993) [subfamily

I.1 as defined by Eltis and Bolin (Eltis and Bolin, 1996)]

and may support the metabolism of chlorinated biphenyl

congeners (McKay et al., 2003; Fortin et al., 2005)

(Fig. 2). Even beyond the well-documented vicinal chelate

superfamily, 2,3-dihydroxybiphenyl dioxygenases have

been documented. As an example, BphC6 of Rhodococ-

cus jostii RHA1 (ABO34703) or BphC3 of Rhodococcus

rhodochrous K37 (Taguchi et al., 2004) belong to the

so-called LigB family (Sugimoto et al., 1999), members

of which are well recognized as being responsible for

the degradation of protocatechuate via the protocat-

echuate 4,5-dioxygenase pathway or of cleaving 2,3-

dihydroxyphenylpropionate (Spence et al., 1996) or

2-aminophenol (Takenaka et al., 2000). Additional LigB

type enzymes have been described to be involved in the

degradation of bi- and polycyclic aromatics (Laurie and

LloydJones, 1999; Gibbs et al., 2003); however, respec-

tive genes are not typically targeted in environmentalsurveys. In contrast, catechol 1,2-dioxygenases have

been proposed as markers for aromatic degradative

potential (Cavalca et al., 2004). Although this seems

logical to some extent, it must be considered that genome

sequencing projects are revealing that respective genes

belong to the core genome of Burkholderia as well as a

large subset of Pseudomonas species (Perez-Pantoja

et al., 2009) and may indicate the fitness of the respective

hosts rather than selection of respective catabolic genes.

Aerobic alkane degradation

The degradation of alkanes has been for a long time

associated with the presence of an AlkB integral-

membrane non-haem diiron monooxygenase as is the

case for P. putida GPo1 (van Beilen et al., 1994). Since

then, alkane monooxygenases have been observed in

various Proteobacteria and in Actinomycetales (van

Beilen and Funhoff, 2007) and the growing collection of

alkane hydroxylase gene sequences has allowed the

analysis of their diversity and abundance in different envi-

ronmental systems (Hamamura et al., 2008; Wasmund

et al., 2009). The quantity of alkB genes has been found

to be correlated with n-alkane concentrations in petroleum

contaminated soils (Powell et al., 2006). However, recent

reports show that the terminal oxidation of alkanes can

also be catalysed by completely distinct enzyme systems.

In 2001, the first bacterial cytochrome P450-dependent

alkane monooxygenase was described from Acineto-

bacter sp. EB104 and termed Cyp153A1 (Maier et al.,2001). In the meantime, genes encoding cytochrome

P450 CYP153 family proteins have been detected in a

broad set of bacterial genera such as Mycobacterium

(Funhoff et al., 2006) and Alcanivorax previously

described to harbour AlkB encoding genes (van Beilen

et al., 2004) as well as in genera not previously reported

to be oil degraders such as Idiomarina or Erythrobacter

(Wang et al., 2010) and are specifically common in

alkane-degrading eubacteria lacking AlkB encoding

genes (van Beilen et al., 2006). While their environmental

importance has yet to be assessed in detail, some

CYP153-encoding gene fragments have already been

isolated from different environments and chimeric genes

encoding functional proteins could successfully be

created (Kubota et al., 2005).

Until recently, very limited information was available on

the degradation of long-chain alkanes. In Acinetobacter

sp. DSM 17874, able to grown on alkanes with chain

lengths of up to 40 C atoms, a flavin-binding monooxyge-

nase encoded by almA was identified as being involved in

the metabolism of long-chain alkanes (Throne-Holst et al.,

2007). Even though homologues were identified in

various Acinetobacterstrains, including Acinetobactersp.

M-1, where such activity was observed for the first time

(Maeng et al., 1996), nothing is known about the environ-mental distribution of this gene type in contaminated sites.

The same holds for LadA proteins. LadA is a flavoprotein

monooxygenase that initiates the degradation of C15C36

alkanes in Geobacillus thermodenitrificansNG80-2 (Feng

et al., 2007) and has recently been shown to be a member

of the Ssu subfamily of the bacterial luciferase family (Li

et al., 2008). Clearly, attempts to characterize the cata-

bolic diversity and functions involved in alkane degrada-

tion at contaminated environments have to take into




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consideration the high diversity of enzymes capable of

initiating such metabolism.

Fine-scale diversity

The high diversity of enzymes and catabolic routes crucial

for bacterial metabolism of pollutants is not the only chal-

lenge we face with when performing molecular diagnos-

tics of polluted environments. It is well documented that

single amino acid differences may have drastic influences

on enzyme properties. As an example, the Rieske non-

haem iron oxygenases are a large superfamily and have

been further classified into subfamilies where typically,

members of a subfamily share similarities in substrate

specificity (Gibson and Parales, 2000). However, single

amino acid differences may influence the regioselectivity

and enantioselectivity of hydroxylation, as exemplified by

naphthalene dioxygenase mediated attack on biphenyl or

phenanthrene (Parales et al., 2000). Depending on the

mode of hydroxylation, the substrate may be channelledinto a productive route resulting in mineralization or the

substrate may be co-metabolized resulting in the forma-

tion of dead-end products or intermediates that may

further be catabolized by other community members

present at the contaminated site. Such misrouting is most

evident when comparing metabolic routes for, e.g. biphe-

nyl and aromatic biarylethers such as dibenzofuran. While

biphenyl is typically mineralized after 1,2-dioxygenation

(so-called lateral dioxygenation) (Pieper and Seeger,

2008), dibenzofuran which may be regarded as a doubly

ortho-substituted biphenyl requires attack at the quasi

ortho carbon (the angular position) and its neighbour

(Fig. 3) to cleave the ether-bond (Armengaud et al.,

1998), and lateral dioxygenation results in the formation of

dead-end products. Single crucial amino acid differences

were also reported to significantly change the substrate

range and substitution of a methionine by alanine in

toluene dioxygenase enabled the enzyme to transform

tetrachlorobenzene, probably by facilitating access of the

voluminous substrate tetrachlorobenzene to the active-

site iron (Beil et al., 1998). Thus, to obtain an overview of

the catabolic potential of contaminated sites, it is impor-

tant not only to analyse the relative quantities of catabolic

gene groups but also their diversity. As an example, a

survey of a benzene contaminated site targeting the

toluene/isopropylbenzene/biphenyl subfamily of Rieske

non-haem iron oxygenases revealed the predominance of

gene fragments, which are similar to those encoding iso-

propylbenzene dioxygenases. However, modelling of the

active site and analysis of isolates harbouring respective

genes revealed one of the predominant genes to harbour

voluminous methionine residues at the active site, which

has been proposed to prevent access of toluene (and

isopropylbenzene) to the active site and thus the failure of

respective isolates to grow on toluene (and also on iso-

propylbenzene) (Witzig et al., 2006).

Tools to analyse catabolic gene diversity

The detection of functional genes is usually performed

through the analysis of clone libraries of gene fragments

amplified using primers targeting a given gene family or

through different DNA fingerprinting methods such as ter-

minal restriction fragment length polymorphism (T-RFLP)

(Sipila et al., 2008), denaturing or temperature gradient

gel electrophoresis (DGGE/TGGE) (Gomes et al., 2007)

or single strand conformation polymorphism (SSCP)

(Junca and Pieper, 2004), with the last two mentioned

methods giving direct access to sequence information. To

achieve the simultaneous detection of multiple genes,

microarrays consisting of probes of PCR fragments

derived from reference genes or oligonucleotides and

Fig. 3. Different regioselectivities of dioxygenolytic attack observed with phenanthrene as substrate (left) and lateral versus angulardioxygenation observed with dibenzofuran as substrate.




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designed to anneal to sequences representing different

catabolic gene families have been developed in the last

decade. The advantage of such array systems is the

amount of different sequences that can be detected in a

single assay, contrasting PCR primer-based detections,

where usually only a subset of a catabolic gene family can

be targeted with a single primer set. However, arrays

require time for careful design, are relatively costly and

require detailed processing of information. The obtained

results also require validation to confirm the correctness

of signals.

An oligoarray to detect hundreds of functions related to

bacterial degradation of pollutants, including catabolic,

regulatory, resistance and stress genes, has been

reported (Rhee et al., 2004) and evolved as the so-called

GeoChip (He et al., 2007). There are some additional

interesting approaches in the field of microarrays to detect

catabolic functions related to aerobic aromatic biodegra-

dation, such as the oligoarrays that specifically target

Rieske non-haem iron oxygenases or monooxygenases(Iwai et al., 2008). However, at the present state, optimiz-

ing functional gene arrays is still necessary, as appropri-

ate standards for data comparison and normalization are

lacking and comparisons between microarray data across

different sites, experiments and time periods is difficult

(Liang et al., 2010).

New high-throughput sequencing technologies such as

the 454 GS FLX (Roche), or the Genome Analyser (Illu-

mina) will also change approaches for assessing cata-

bolic gene diversity as in theory, a high number of PCR

amplification products can be directly subject to sequenc-

ing. Even though such approaches are so far typically

used to analyse community structure by sequencing of

16S rDNA amplicons (Liu et al., 2007; Roesch et al.,

2007; Lazarevic et al., 2009), amplicon pyrosequencing

has already been employed to target the diversity of

biphenyl dioxygenases of the Rieske non-haem iron oxy-

genase superfamily (Iwai et al., 2010).

Function-based screening for novel activities

As stated above, new metabolic and enzymatic mecha-

nisms involved in pollutant degradation are still being

discovered. Even primer-based approaches, designed

based on known metabolic diversity and on describedmechanisms, are uncovering a broader diversity of

enzymes than previously thought. The real microbial cata-

bolic diversity of the environment is still awaiting to be

deciphered.

Recent progress has revealed that the capture of

genetic resources of complex microbial communities in

metagenome libraries allows the discovery of a richness

of new genetic diversity that had not previously been

imagined (Ferrer et al., 2005; Beloqui et al., 2006).

However, only a few reports clearly attempted to identify

catabolic genes directly from environmental DNA by a

metagenomic approach. Using the yellow coloration of

catechol ring-cleavage products as functional screen,

Brennerova and colleagues (2009) targeted a BTEX-

contaminated environment and could identify one cat-

echol extradiol dioxygenase activity to be encoded per

3.6 Mb of DNA screened from a fosmid library constructed

in Escherichia coli, indicating a massively high abundance

of these genes at the site. Interestingly, only one-fourth of

the observed extradiol dioxygenases belonged to subfam-

ily I.3.A or I.3.B (see Fig. 2) that would be expected as

predominant taking into consideration the knowledge

gained from isolates. Genes of subfamily I.2.A were

absent, but a high abundance of genes with similarity to

DbtC of Burkholderia sp. DBT1 (Di Gregorio et al., 2004)

was observed. Based on specificity constants of enzymes

expressed from the fosmids, a task-sharing between dif-

ferent extradiol dioxygenases in the community of the

contaminated site can be supposed, attaining a comple-mentary and community-balanced catalytic power against

diverse catecholic derivatives, as necessary for effective

degradation of mixtures of aromatics.

Also Suenaga and colleagues (2007) used a function-

driven metagenomic approach to screen environmental

DNA prepared from an active sludge used to treat coke

plant wastewater. Even though extradiol dioxygenases

typically observed in Proteobacteria such as enzymes of

subfamily I.2.A were observed, the library was dominated

by clones harbouring extradiol dioxygenases with homol-

ogy to the manganese dependent 2,3-dihydroxybiphenyl

dioxygenase of Bacillus sp. JF8 (Hatta et al., 2003)

(Fig. 2), which, however, preferred catechol over 2,3-

dihydroxybiphenyl as a substrate (Suenaga et al., 2009a).

In addition, the library contained clones with extradiol

dioxygenases having homology to BphC 2,3-

dihydroxybiphenyl dioxygenase of Terrabacter sp.

DPO360 (Schmid et al., 1997) (Fig. 2) indicating Firmic-

utes and Actinobacteria to be important for biodegrada-

tion by the sludge. Sequencing revealed that only a

subset of clones contained complete degradation path-

ways whereas the majority of clones contained a subset

of pathway genes in novel gene rearrangements

(Suenaga et al., 2009b). However, the fact that aromatic

compounds in the environment may be degraded throughthe concerted action of various fragmented pathways has

also been supported by the study on isolates. As an

example P. putida GJ31 degrades chlorobenzene by

activities recruited from four different pathway modules

(Kunze et al., 2009). Importantly, even though genes

encoding a multicomponent phenol hydroxylase (Shingler

et al., 1989), typically used as a target to characterize the

potential and diversity for phenol degradation (Watanabe

et al., 1998), have been observed on one fosmid, a gene




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encoding a single component phenol hydroxylase with

similarity to that identified from Geobacillus stearothermo-

philusBR219 (Kim and Oriel, 1995) was observed in high

abundance, indicating bacilli to be important for phenol

degradation in the sludge.

It is known that several types of oxygenases when

expressed in E. coli are able to produce the blue pigment

indigo via the oxidation of indole, which is formed from

tryptophan by E. colitryptophanase. Indigo formation was

then used to functionally screen a metagenomic library

resulting in the discovery of a styrene oxidase only dis-

tantly related to those that have been previously charac-

terized (van Hellemond et al., 2007). However, there are

many biotransformation processes of interest that do not

produce metabolites that can be easily detected by simple

activity tests (such as reaction colour). Moreover, metage-

nomic library clones are usually in numbers that are not

suitable for single chemical analyses. Thus, alternative

high-throughput methods to screen such kind of libraries

are needed. One approach to overcome these limitationsuses a transcriptional regulator that is blind to the reaction

substrate but responds to the reaction product, and as a

result activates a promoter fused to a reporter gene

(Galvao and de Lorenzo, 2006). Respective regulators

may be searched for in natural regulatory circuits, but can

also be engineered in order to recognize the product of

the desired activity. Bacteria containing such a regulator/

promoter/reporter system may then be used as receptors

of a metagenomic library and only the clones hosting a

metagenomic insert encoding an enzyme capable of

catalysing the desired reaction should activate the

reporter gene. Respective genetic traps have recently

been established for translating the transformation of

gamma-hexachlorocyclohexane (HCH) into detectable

signals by using a regulator responsive to 1,2,4-

trichlorobenzene, a major product of HCH dehydrochlori-

nation (Mohn et al., 2006). Another approach is based on

the knowledge that catabolic gene expression is typically

induced by relevant substrates and, in many cases, con-

trolled by regulatory elements situated in proximity to

catabolic genes. Random cloning of environmental DNA

in front of a promoterless green fluorescent protein (GFP)

reporter followed by fluorescence-activated cell sorting

enrichment of the expression pool in the presence of the

target substrates benzoate and naphthalene was thenused to select for clones that bear catalytic activities

related to the substrate (Uchiyama et al., 2005). In fact,

benzoate catabolic genes could be observed by this

approach. However, it was also discussed that this

approach is not without problems as transcriptional regu-

lators might be activated by effectors that are not sub-

strates of the pathways they regulate and may, thus,

endow the system with considerable noise of false posi-

tives (Galvao and de Lorenzo, 2006).

Mining bacterial genomes

Those molecular techniques described above enable us

to directly extract and express novel information directly

from contaminated sites irrespective of whether the hosts

are cultivable or not. However, not only metagenomic but

also genomic analyses of single strains constitute an

immense source for discovering and exploiting novel bio-

catalysts. In general, genomic information of sequenced

microorganisms can be used in at least two levels, on the

one hand to elucidate genes where the function of

encoded enzymes is unknown and on the other to better

understand the metabolic network of strains endowed

with a broad catabolic diversity. At present, 1247 bacterial

genome sequences are listed at http://www.ncbi.nlm.

nih.gov/sutils/genom_table.cgi and 907 finished and 838

draft sequences at http://img.jgi.doe.gov/cgi-bin/pub/

main.cgi, comprising the complete genomes of biode-

grading bacteria such as Burkholderia xenovoransLB400

(Chain et al., 2006), R. jostiiRHA1 (McLeod et al., 2006),Cupriavidus necatorJMP 134 (Perez-Pantoja et al., 2008;

Lykidis et al., 2010), P. putida KT2440 (Nelson et al.,

2002) or Mycobacterium vanbaalenii PYR-1 (Kim et al.,

2008) and the genomic backgrounds for their abilities to

utilize certain pollutants have been revealed. A detailed

metabolic reconstruction has been performed using C.

necator JMP134 to develop a detailed overview of its

metabolism from an analysis of the genome sequence

(Perez-Pantoja et al., 2008) and to link the catabolic abili-

ties predicted in silico with the range of compounds that

support growth of this bacterium. Of the 140 aromatic

compounds tested, 60 serve as a sole carbon and energy

source for this strain, strongly correlating with those cata-

bolic abilities predicted from genomic data. However, the

more interesting cases are where in silicopredictions and

experimental results do not fit.

At the gene level, information is available on the deg-

radation of 4-hydroxyphenylacetate through monooxy-

genation by a two-component 4-hydroxyphenylacetate

hydroxylase and homoprotocatechuate as central

intermediate (Prieto and Garcia, 1994). However,

JMP134 does not harbour genes encoding respective

activities (Perez-Pantoja et al., 2008). In contrast,

4-hydroxyphenylacetate is likely to be metabolized by the

homogentisate pathway in C. necator, thus involvinghydroxylation of the aromatic ring at C-1 with a concomi-

tant migration of the carboxymethyl side chain to C-2 (the

NIH shift reaction), catalysed by a NADH-dependent

4-hydroxyphenylacetate-1-hydroxylase, for which, how-

ever, no sequence data are available (Hareland et al.,

1975) (see Fig. 1).

In the course of a genomic in silico search for novel

aromatic ring-cleavage dioxygenases in P. putidaKT2440,

a gene could be identified, the product of which showed


http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgihttp://www.ncbi.nlm.nih.gov/sutils/genom_table.cgihttp://img.jgi.doe.gov/cgi-bin/pub/main.cgihttp://img.jgi.doe.gov/cgi-bin/pub/main.cgihttp://img.jgi.doe.gov/cgi-bin/pub/main.cgihttp://img.jgi.doe.gov/cgi-bin/pub/main.cgihttp://www.ncbi.nlm.nih.gov/sutils/genom_table.cgihttp://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi


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significant similarity to protocatechuate 4,5-dioxygenases,

suggesting that it could be involved in the metacleavage of

a catecholic compound, a type of reaction that had not

been reported yet in P. putida KT2440 (Nogales et al.,

2005). Substrate screening of the overexpressed extradiol

dioxygenase identified it as a gallate dioxygenase (Fig. 1)

being the prototype of a new subgroup of type II extradiol

dioxygenases that shares a common ancestor with proto-

catechuate 4,5-dioxygenases and whose two-domain

architecturemight have evolved from thefusionof thelarge

and small subunits of the latter. Gallate dioxygenases were

recently identified in 22 out of 822 genomes analysed

for the distribution of aromatic catabolic properties,

being as abundantly distributed as protocatechuate 4,5-

dioxygenases (Perez-Pantoja et al., 2009). However, the

respective genes are typically annotated as protocat-

echuate 4,5-dioxygenases, even though at least the P.

putidaKT2440 gene product does not exhibit such activity.

Genome in silicoanalysis also led to the identification of

a gene cluster involved in nicotinic acid degradation in P.putida KT2440 (Jimenez et al., 2008) being the first com-

plete set of genes identified encoding degradation of this

compound. Also, novel knowledge on the degradation of

gentisate, a key intermediate in the degradation of many

aromatic compounds such as salicylate or 3-hydroxyben-

zoate, could be generated through genome mining. In the

gentisate pathway, gentisate 1,2-dioxygenase, a member

of thecupin superfamily, cleaves thearomaticring between

the carboxyl substituent and theproximal hydroxyl group to

yield maleylpyruvate (Crawford et al., 1975). Isomerization

of maleylpyruvate to fumarylpyruvate is catalysed by

either a glutathione (GSH)-dependent maleylpyruvate

isomerase almost exclusively found in Gram-negative bac-

teria (Crawford et al., 1975), or a GSH-independent maley-

lpyruvate isomerase that has been characterized in

various Gram-positive bacteria (Crawfordand Frick, 1977).

Mining the genome of Corynebacterium glutamicum

resulted in the first identification of genes involved in the

GSH-independent pathway, which were observed to be

encoded in the same catabolic gene cluster as is gentisate

dioxygenase (Shen et al., 2005). Genome mining to dis-

cover and exploit novel enzymes also targeted, among

others, BaeyerVilliger monooxygenases (BVMOs),

leading to the discovery of the first thermostable enzyme

of this group (Fraaije et al., 2005). Interestingly, thesequenced genome of R. jostiiRHA1 encoded 23 putative

BVMOs out of which 13 could be heterologously expressed

showing a remarkable diversity of both regio- and enanti-

oselectivity (Szolkowy et al., 2009).

Insights into the metabolism at the

organism-wide level

Genomics studies could allow a reconstruction of meta-

bolic pathways relevant for biodegradation of xenobiotics,

providing a holistic (or systems) view on the metabolic

network of a particular organism. Quite importantly,

among current bottlenecks in genome analysis the lack of

knowledge and insufficient efforts on enzymology and

amplifying annotation mistakes in databases are of great-

est hindrance for functional reconstruction.

Overall, it is evident that a large proportion of the ORFs

of newly sequenced genomes have little sequence homol-

ogy with known enzymes, so their potential activities

remain hidden. There is, however, an increasing number

of methods for predicting protein function from sequence

or structural data (for a recent review, see Lee et al.,

2007). Even though annotation strategies have become

more sophisticated in recent years (Rentzsch and

Orengo, 2009), it needs to be noted that the majority of

protein sequences in public databases have not been

experimentally characterized and the most common

approach in use today continues to be the assignment of

molecular function from the inference of homology fol-

lowed by annotation transfer (Schnoes et al., 2009).Recently, the misannotation levels for molecular function

in public protein sequence databases was investigated for

a model set of 37 enzyme families for which extensive

experimental information was available (Schnoes et al.,

2009). The authors observed surprisingly high levels of

misannotation of up to > 80% for some of the subfamilies

studied, mainly associated with overprediction of

molecular function and an increase in misannotations

from 1993 to 2005. Thus, they stated that misannotation in

enzyme superfamilies containing multiple families that

catalyse different reactions is a larger problem than has

been recognized. The same problem holds also when

considering aromatic degradation reactions. As an

example, gallate dioxygenases mentioned above as

observed in 22 out of > 800 genome sequencing pro-

jects are typically annotated as protocatechuate 4,5-

dioxygenases. A phylogenomic approach was recently

used to analyse for the presence of aromatic degradative

pathway in sequenced genomes (Perez-Pantoja et al.,

2009) which provides clues on the distribution of catabolic

properties among bacterial phyla and on the ecological

functions of specific bacterial groups, defines under-

scored research objectives and gives a better overview of

the genetic basis of bacterial catabolism of aromatics. The

phylogenomic approach to study the organization of aro-matic degradation was based on the selection of

sequences of key catabolic functions derived from both

biochemically and genetically well-studied systems to fish

into the sequenced genome databases, followed by

refinement of the positive scores and identified a huge set

of misannotations in public databases.

The whole-genome sequences have not only enabled

us to identify genes and to predict (and possibly confirm)

their biological roles through homologous comparisons,




10/16

but to also identify novel biocatalysts. In concert with

proteomic and transcriptomic information, new insights

into the metabolism at the organism-wide level could be

obtained. As an example, metabolic, genomic and pro-

teomic approaches were used to construct a complete

and integrated pathway for pyrene degradation in M. van-

baaleniiPYR-1 (Kim et al., 2007). However, not only could

the metabolic pathway be defined and proteins involved

identified, but also differences in the metabolism of differ-

ent PAHs revealed and thus suggestions for the involve-

ment of additional candidate genes in the complex

network of PAH (polycyclic aromatic hydrocarbon)

metabolism made (Kim et al., 2008). Moreover, a large

number of determinants associated with protection

against PAH substrates and metabolites were observed,

comprising, for example, a catechol-O-methyltransferase

(Kim et al., 2008). Putative detoxification mechanisms

were also revealed during analysis of the transcriptome of

B. xenovorans LB400 (Parnell et al., 2006). Transcrip-

tomic and proteomic studies also revealed insights intothe role of benzoate-catabolic pathway redundancy with

the so-called box-pathway responsible for degradation of

benzoate via benzoyl-CoA being preferentially expressed

under reduced oxygen concentrations, thus, relating this

redundancy to possible adaptations to different environ-

mental conditions (Denef et al., 2005; 2006) and capabili-

ties of bacteria to deal with oxidative stress generated

during the metabolism of aromatics (Agullo et al., 2007).

Progress has also now been made to unravel and under-

stand full bacterial genome regulatory networks and pol-

lutant physiology under conditions of environmental

stresses, to suggest experimental ways for limiting stress

effects while maintaining bacteria catabolic efficiency. As

an example, fluctuation in water availability is a funda-

mental stress challenging soil-residing microorganisms,

and desiccation tolerance is a key adaptation of many

such organisms. Factors contributing to the desiccation

resistance in the versatile biodegrader R. jostii RHA1

were recently identified, comprising the biosynthetic

pathway of a compatible solute (LeBlanc et al., 2008).

Synthesis of compatible solutes, protection from oxidative

damage, transcriptional regulation and cell envelope

modification seem to be common mechanisms to deal

with desiccation stress (Katoh et al., 2004; Cytryn et al.,

2007).To fully understand how bacteria respond to their envi-

ronment, it is clearly essential to assess genome-wide

transcriptional activity. New high-throughput sequencing

technologies such as the 454 GS FLX (Roche) or the

Genome Analyser (Illumina) make it possible to query the

transcriptome of an organism in an efficient unbiased

manner (Sorek and Cossart, 2010). This method termed

RNA-Seq (RNA sequencing, or better sequencing of

cDNA fragments) has initially been applied to the analysis

of eukaryotic transcriptomes (Wang et al., 2009). In fact,

mRNA enrichment is more challenging in prokaryotes, as

prokaryotic mRNAs lack the 3-end poly(A) tail of mRNAs

in eukaryotes and as the majority of cellular RNA is com-

posed of ribosomal RNA and tRNA, such that transcrip-

tome sequencing of non-enriched total RNA would yield

mostly non-mRNA sequences (Sorek and Cossart, 2010).

Recently, with the application of methods such as the

artificial polyadenylation of mRNA (Frias-Lopez et al.,

2008) and the depletion of processed RNA (rRNA and

tRNA), RNA-Seq has been extended to the study of

microbes (Yoder-Himes et al., 2009; Filiatrault et al.,

2010). Importantly, all of these studies show that the bac-

terial transcriptome is significantly more complex than

previously thought and revealed the presence of a huge

set of non-coding RNAs (ncRNAs), novel untranslated

regulatory elements and alternative operon structures

(Sorek and Cossart, 2010).

On-site catabolic gene expression

As the detection of functional genes provides information

on the presence of organisms harbouring the respective

genes at a site and possibly on a selective advantage for

the host to harbour such catabolic genes, functional gene

abundance does not directly reflect metabolic activity. To

document expression of specific genes, analysis of

mRNA is applied. As with pure culture studies, initial

studies typically concentrated on documenting transcrip-

tion of specific target genes such as naphthalene dioxy-

genase encoding genes (Wilson et al., 1999; Yagi and

Madsen, 2009). Until recently, the mRNA approach was

hampered by low yields of mRNA retrieved from environ-

mental samples and its rapid decay. However, novel

methodological developments such as those described

above for pure culture studies, and specifically the

T7-RNA-polymerase-based RNA amplification (originally

introduced by Van Gelder et al., 1990) where A-tailed

RNA is reverse-transcribed primed with an oligo(dT)

primer containing a T7 promoter sequence allowing a

1000-fold unbiased amplification makes metatrancrip-

tomic studies now feasible (Frias-Lopez et al., 2008).

Optimized mRNA extraction, purification and amplification

protocols were recently used to analyse a small library of

cDNA clones of a crude oil-degrading marine microbialcommunity making evident not only the expected expres-

sion of genes related to the biodegradation of fatty acids

but also of those involved in the biosynthesis of glycolipids

probably involved in emulsification of crude oil (Kato and

Watanabe, 2009). They were also used in concert with

pyrosequencing to analyse complex microbial communi-

ties such as the oceans water column revealing, among

others an impressive array of novel ncRNAs, some of

which were suggested as regulators for carbon metabo-




11/16

lism and energy production (Shi et al., 2009). Clearly

metatranscriptomic analyses will improve our knowledge

on the expressed subset of metagenomic DNA and on

functioning of and interactions among members of micro-

bial communities. New sequencing technologies, such as

the upcoming nucleic acids single true molecule sequenc-

ing, especially those non-fluorescent or Raman-based

methods (Treffer and Deckert, 2010), will, without doubt,

not only allow the analysis of larger fractions of the

metagenome and metatranscriptome, but also, for

example in parallel with microarray analyses or gene

family-targeted mRNA pyrosequencing, a better under-

standing of the functional interactions in biodegradation

and bioremediation.

Concluding remarks

Any rational effort to interfere with microbial processes in

order to optimize metabolic performance on site has to

deal with the enormous complexity of the system. For-tunately, new technological developments and concep-

tual frameworks provide new approaches to explore

complex biological settings, allowing us to move towards

a picture of the complete catalytic potential and the

metabolic net of the bacterial communities that thrive in

polluted sites.

The speed and depth by which ecosystem functioning

can be described is heavily influenced by new technical

developments. Specifically in DNA sequencing technolo-

gies, the impact of 454, Illumina and the forthcoming

single-molecule sequencing platforms will change once

more the scale and depth of explorations of microbial

communities. Some approaches that were previously

technically impossible are now plausible, such as obtain-

ing the complete genome sequence of a single bacterial

cell, by using cell separation methods (Vives-Rego et al.,

2000) and isothermal amplifications of the genomic

DNA contained in one single cell (Woyke et al., 2009). The

single bacterial cell analysis is also being developed for

other cellular components such as metabolites and pro-

teins (Burg et al., 2007; Borland et al., 2008) and we can

foresee its application to analyse catabolic potential or

activity against aromatics of specific cell groups from

microbial communities in bioremediation treatments.

Technical advances in metabolomics (Hirai et al., 2004;Giavalisco et al., 2008; Iijima et al., 2008) allow the cor-

relation with expression profiles and, of course, genomic

content (Hirai et al., 2005). The potential of these metabo-

lomic analyses can be foreseen when adapted to assess

microbial community biodegradation performance, inte-

grating more and more biological processes, for example,

analyses of metabolite fluxes, of pathway bottlenecks,

determine how communities cope with stress and how

they adapt to changing environments.

To achieve a closer description of the catabolic network

and its components and to gain the potential for modelling

of the environmental selectors to be able to predict eco-

system behaviour, it is necessary to integrate experimen-

tal information in a high-throughput manner (Trigo et al.,

2009). In fact, the huge set of information collected from

the analysis of different descriptors of microbial commu-

nity functioning will require novel ways to organize data

and extract meaningful conclusions. There is an urgent

need to build expanded custom metabolic networks cov-

ering all described pathways for target pollutants. This will

require a carefully curated framework to define catabolic

genes in a much more precise way than the current auto-

matic genomic annotation. Community descriptions will

also require the use of tools from the nascent systems

biology field (Fisher and Piterman, 2010; Gehlenborg

et al., 2010; Liu et al., 2010) which rely on computational

biology and visualization tools to be able to define the

phylogenetic composition and shifts, the functions

selected or expressed, their association with certainmetabolic steps, the metabolites fluxes and the compari-

son of the observed patterns between samples.

Hydrocarbon contamination in environmental setups

may be regarded as a large evolutionary metabolic model

suited to study the effects of strong selectors on complex

microbial populations and the catabolic landscape (de

Lorenzo, 2008). We do not have yet enough experiments

comparing, under controlled conditions, the catabolic/

taxonomic or network responses of samples from diverse

biogeographic origins challenged by the same pollution or

selector. By analysing the ecology of biodegradation, we

may add experimental information on how from the pro-

posed homogenous and ubiquitous presence of all bac-

terial types (De Wit and Bouvier, 2006) the apparently

extremely high diversity of bacterial community composi-

tion on Earth developed (Sogin et al., 2006). Such analy-

sis will help us to understand basic aspects of functional

selection and microbial diversity, and how predictable

such behaviour is, dependent on the origin of a site to

bioremediate, and dependent on the abiotic factors quan-

tified. Such systems understanding will open new ways to

improve sustainable use of our environment.

AcknowledgementsWe would like to thank former and current members of the

Microbial Interactions and Processes Research Group, HZI

Helmholtz Centre for Infection Research (previously known

as AG Biodegradation at the German Research Centre for

Biotechnology, Braunschweig) for all their help and support

during the last years, in our quest to improve the understand-

ing of the ecology of microbial aromatic biodegradation.

Research within the authors laboratories was funded by the

projects BIOTOOL, BACSIN and MAGICPAH from the Euro-

pean Commission.




12/16

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