Monitoring biodegradative enzymes with nanobodies raised in Camelus dromedarius with mixtures of...

Monitoring biodegradative enzymes with nanobodiesraised in Camelus dromedarius with mixtures ofcatabolic proteinsemi_2401 1..15

Olga Zafra,1† Sofía Fraile,1 Carlos Gutiérrez,2

Amparo Haro,1‡ A. David Páez-Espino,1§

Jose I. Jiménez1¶ and Víctor de Lorenzo1*1Systems Biology Program. Centro Nacional deBiotecnología-CSIC, Campus de Cantoblanco, 28049Madrid, Spain.2Department of Animal Pathology, Faculty of VeterinaryScience, Las Palmas University, 35416 Canary Islands,Spain.

Summary

Functional studies of biodegradative activities inenvironmental microorganisms require moleculartools for monitoring catabolic enzymes in themembers of the native microbiota. To this end, wehave generated repertories of single-domain VHH frag-ments of camel immunoglobulins (nanobodies) ableto interact with multiple proteins that are descriptorsof environmentally relevant processes. For this, weimmunized Camelus dromedarius with a cocktail ofup to 12 purified enzymes that are representativeof major types of detoxifying activities found inaerobic and anaerobic microorganisms. Followingthe capture of the antigen-binding modules from themRNA of the camel lymphocytes and the selection ofsub-libraries for each of the enzymes in a phagedisplay system we found a large number of VHH

modules that interacted with each of the antigens.Those associated to the enzyme 2,3 dihydroxybiphe-nyl dioxygenase of Burkholderia xenovorans LB400(BphC) and the arsenate reductase of Staphylococ-cus aureus (ArsC) were examined in detail and foundto hold different qualities that were optimal for dis-tinct protein recognition procedures. The repertory ofanti-BphC VHHs included variants with a strong affinityand specificity for linear epitopes of the enzyme.

When the anti-BphC VHH library was recloned in aprokaryotic intracellular expression system, somenanobodies were found to inhibit the dioxygenaseactivity in vivo. Furthermore, anti-ArsC VHHs were ableto discriminate between proteins stemming from dif-ferent enzyme families. The easiness of generatinglarge collections of binders with different propertieswidens considerably the molecular toolbox for analy-sis of biodegradative bacteria and opens fresh possi-bilities of monitoring protein markers and activities inthe environment.

Introduction

The effect of pollutants in the environment is a longstand-ing and recurrent matter of concern for both the scientificcommunity and the general public (Gomez et al., 2007;Kolok and Sellin, 2008; Kümmerer, 2009a,b; Liu et al.,2009; Bombach et al., 2010). Various schemes have beendeveloped for rapid assessment of ecological impact ofchemical waste, e.g. biotoxicity (Leitgib et al., 2007) andbioavailability (Belkin, 2003; van der Meer and Belkin,2010) by means of different biosensors and bioindicators(Tecon et al., 2010). Many contaminants are xenobioticcompounds, which frequently become the substrates ofmicrobial modifications (Parales et al., 2002). Their fateand that of their derivatives in polluted sites have thusraised a considerable interest, and several platforms forpredicting the ultimate destination of chemicals uponexposure to the global microbial metabolism have beenproposed (Gomez et al., 2007; Wicker et al., 2010). Buthow to match predicted metabolic capacities with actualoccurrence of biodegradative enzymes?

One criterion for assessing the biodegradative potentialof a polluted site is the enumeration of resident microbialspecies as well as the abundance and diversity of theircatabolic genes (He et al., 2010). This is based on thenotion that availability of new C and N sources broughtabout by given pollutants will favour those species thatboth endure the corresponding stress and can metabolizesuch compounds (de Lorenzo, 2008). DNA chip technolo-gy (He et al., 2010) has also been used to tackle suchenvironmental diagnose problem. In this work we haveexplored an alternative approach to the same end,

Received 7 July, 2010; accepted 12 November, 2010. *For correspon-dence. E-mail [email protected]; Tel. (+34) 91 585 45 36; Fax(+34) 91 585 45 06. Present addresses: †Centro de Astrobiología,INTA-CSIC, 28850 Torrejón de Ardoz, Spain; ‡Ministerio del MedioAmbiente, Department. Environmental Quality, 28003 Madrid, Spain;§Geomicrobiology Program, University of California, Berkeley, CA94720, USA; ¶Harvard FAS Center for Systems Biology, Cambridge,MA 02138, USA.

Environmental Microbiology (2011) doi:10.1111/j.1462-2920.2010.02401.x

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd

namely the use of recombinant antibodies for recognitionof key enzymes of environmental significance throughimmunological tests. The main issue is the implementa-tion of a reliable workflow for preparation of such antibod-ies in numbers and quantities that circumvent problems ofproduction time and scale, low yields or low diversity thatrender only a few clones likely to be used. In this context,we report below the generation of a library of recombinantcamel antibodies against a cocktail of purified proteinswith important roles in environmental biochemistry, andtheir eventual production and isolation in various prokary-otic expression systems. Features of the library that areworth of note include its diversity, specificity and sensitiv-ity. Furthermore, each antibody pool contained specimensof VHH domains (nanobodies; Harmsen and De Haard,2007) optimized for identification of the antigens in bothnative and denatured conformations with various specific-ity levels in vivo and in vitro. The power of such a platformis demonstrated for answering a number of standingquestions on the regulation of the duplicated arsenic-resistance operons in Pseudomonas putida (Cánovaset al., 2003) and to examine the problem of gratuitousinduction of the bph genes for biphenyl biodegradation byBurkholderia xenovorans LB400 (Beltrametti et al., 2001;Parnell et al., 2009). Furthermore, we show that intracel-lular expression of VHH repertoires against biphenyl 2,3,dioxygenase (BphC) allows the selection of antibodyspecies that block specifically such an enzyme in vivo. Onthese bases, we argue that application of recombinantcamel antibody technology (Hamers-Casterman et al.,1993; Harmsen and De Haard, 2007; Saerens et al.,2008a) to enzymes that are descriptors of archetypicalenvironmental processes results in a fresh moleculardiagnose toolbox for monitoring expression of cataboliccapacities in environmental bacteria.

Results and discussion

Rationale of the production of camel antibody librariesagainst catabolic and stress proteins

The work presented in this article relies entirely on thegeneration of antibody repertoires against a collection ofproteins of environmental interest through immunizationof African camels (Camelus dromedarius) instead of tra-ditional laboratory animals (Harmsen and De Haard,2007). The justification for this choice is that camelidsproduce a distinct type of antibodies composed of onlyheavy chains (Hamers-Casterman et al., 1993), in con-trast with the typical arrangement of heavy (VH) and lightchains (VL) of standard immunoglobulins (Ig). More impor-tant, the complementarity determinant regions (CDRs)that make the actual contacts with the target antigenconsist exclusively of one polypeptide sequence, unlike

the CDRs of ordinary Igs, which are shaped by loops fromboth VH and VL chains. Instead, the antigen-binding part ofcamel antibodies (VHH) consists of a single, comparativelysmall polypeptide (a nanobody; Hamers-Castermanet al., 1993). The preservation of the antigen-bindingproperties in a protein of reduced size endows VHH

modules with unique biophysical properties, e.g. solubility,robustness and ease of folding. These properties makethem most adequate for cloning, heterologous expressionand selection of binders in a prokaryotic phage-displaysystem (De Genst et al., 2006a). An extra bonus of VHH

domains is that the target antigens can be recognized andbound through a protruding paratope in the H3 loop of thestructure. This causes a preference for the recognition ofclefts and crevices in the structure of the immunizedprotein (De Genst et al., 2006b), which often leads to theblockage of the active centre of enzymes (Lauwereyset al., 1998).

Figure 1 summarizes the entire workflow that goes frominoculation of camels with desired antigens to the expres-sion and screening of the corresponding recombinantantibody libraries in a prokaryotic system. Although typicalinoculation protocols for monoclonal antibodies call forimmunization of one animal with one antigen at a time (DeGenst et al., 2006a), camels (as other mammals) mayalso react simultaneously to each of the constituents of acomplex protein mixture (Saerens et al., 2008b). On thisbasis, phage display of a VHH domain library derived froman inoculated animal and panning of the prime poolagainst the separate components of the antigenic mixtureshould allow the capture and amplification of clones spe-cific for each of them (Saerens et al., 2008b).

On the background above, we set out to generateantibody repertoires against 12 purified proteins that arerepresentative of various biological activities of environ-mental interest (Table 1; Fig. 2A). The group includedenzymes of archetypical aerobic biodegradation path-ways (n-alkane monooxygenase, biphenyl dioxygenase,2,3-dihydroxy biphenyl dioxygenase, nitrobenzene dioxy-genase, 2,5-dihydroxypyridine dioxygenase), dehaloge-nation (haloalkane dioxygenase), anaerobic processes(benzylsuccinate synthase and benzoyl-CoA reductase),heavy metal biotransformation (arsenate reductase) andstress markers (glutathione-S-transferase).

Immune reaction of C. dromedarius to environmentallyrelevant proteins

Following inoculation with the 12-enzyme cocktail ofTable 1 (Fig. 2A; see Experimental procedures), theresponse of the animal after a 6 week period was exami-ned by probing a membrane blotted with the proteinsfractionated in a denaturing gel with a dilution of thecorresponding serum (Fig. 2B). Note that detection of

2 O. Zafra et al.

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

antigen-specific antibodies in the Western blot experimentis based on Protein A and it thus detects all bound immu-noglobulins, regardless of their specific type. Further-more, the test reveals only interactions with linearepitopes of the denatured proteins blotted on the mem-brane. The Western blot depicted in Fig. 2B indicated anuneven reaction of the camel to each of the proteins,ranging from quite strong (e.g. ArsC of Sinorhizobiummeliloti, lane 3) to virtually undetectable (for example,LinB of Sphingomonas paucimobilis, lane 5). Not surpris-ingly, in the cases where the protein consisted of a multi-component enzyme (e.g. lane 9, NBDO), one of the bandswas more reactive than the others. To examine in moredetail this immune reaction, we repeated the tests within an enzyme-linked immunosorbent assays (ELISA)system in which target proteins were separately bound tothe wells of a microtitre plate and tested with either theserum of the camel before immunization, or with theserum of the same animal extracted after the whole inocu-lation period (Fig. 2C). Proteins bound to the platesdisplay peptide sequences with various degrees of dena-turation, so it is generally believed that ELISAs reflectmore faithfully the complete immune response than thecorresponding Western blot. The results shown in Fig. 2Cindicated that the animal had a specific reaction to most ofthe proteins of the cocktail, because the pre-immuneserum did not originate any signals worth of note.However, the positive values were not entirely equivalentto those found in the blot of Fig. 2B. On the one hand,some well detectable bands in the Western (e.g. the GSTof Ochrobactrum anthropi, lane 7) gave little signal in theELISA. On the other hand, barely visible – if visible atall – proteins in the blot (e.g. BPDO of Sphingomonasyanoikuyae, lane 10 in Fig. 2B) originated a considerable

reaction in the microtitre wells. In other cases, strongresponses to the same protein were detected with bothprocedures (e.g. ArsC of S. meliloti, lane 3). Only oneprotein (BssA of Thauera aromatica, lane 11) gave poorsignals in both Western and ELISA procedures. For theothers, specific binding to antigens displayed in variousconformations in the ELISA was indicative that the immu-noglobulin pool contained various types of antibodies andthus likely to include those carrying the characteristic VHH

domains that we were after (Omidfar et al., 2004; Rahba-rizadeh et al., 2005). Furthermore, these results validatedthe use of a mixture of proteins rather than individualantigen specimens for generation of the starting materialfor capturing and separating distinct VHH pools for each ofthe products present in the inoculation cocktail. Althoughmixtures of proteins have been used in the past for similarpurposes (Saerens et al., 2008b) we are neither awarethat the number had been as high as in our instance northe immune response as diverse and efficacious as in thecase just presented.

Generation and analysis of VHH libraries

As summarized in Fig. 1, the RNA from lymphocytes iso-lated from the blood of the immunized animal wasextracted, retro-transcribed to DNA and the sequencescorresponding to the VHH domains amplified with specificoligonucleotide primers. The amplified segments werethen captured as fusions to the gIII apical protein of M13and the resulting pool of viral capsides titrated on a sen-sitive E. coli strain (see Experimental procedures sectionfor details). This titre turned out to be ~ 109 of plaque-forming units ml-1 (PFUs), containing a primary diversityof VHHs in the range of 106. This library was separately

Fig. 1. Generation of nanobody libraries workflow. The sketch summarized the main steps of the process. African camels (C. dromedarius)are inoculated with a mixture of proteins and following a 6 week period, RNA from lymphocytes is extracted, retrotrancribed to DNA and thesequences corresponding to the VHH domains amplified with suitable primers for capture in a phage display vector. This library is thenseparately subject to two rounds of panning on plates with the individual antigens of the original inoculation cocktail. Those M13-VHH clonesthat retained a strong binding activity, as detected with a phage-ELISA assay were kept for further analysis.

Nanobodies for biodegradative enzymes 3


Tab

le1.

Pro

tein

sus

edin

the

inoc

ulat

ion

cock

tail.

Pro

tein

sO

rgan

ism

Fun

ctio

nM

w(k

Da)

Ref

eren

ce

Alk

Bp4

50(A

hpG

)M

ycob

acte

rium

sp.

n-al

kane

mon

ooxy

gena

se48

van

Bei

len

etal

.(2

005)

Ars

C(S

m)

Sin

orhi

zobi

umm

elilo

tiA

rsen

ate

redu

ctas

e15

Yan

get

al.

(200

5)A

rsC

(Sa)

Sta

phyl

ococ

cus

aure

usA

rsen

ate

redu

ctas

e16

Mes

sens

etal

.(1

999)

Bss

AT

haue

raar

omat

ica

Ben

zyls

ucci

nate

synt

hase

(asu

buni

t)98

Leut

hner

and

Hei

der

(199

8)B

crA

BC

D(B

zCoA

R)

Tha

uera

arom

atic

aB

enzo

yl-C

oAre

duct

ase

48,

49,

44,

30B

rees

eet

al.

(199

8)B

PD

OS

phin

gom

onas

yano

ikuy

aeB

1B

iphe

nyl-2

,3-

diox

ygen

ase

(oxy

gena

se,

ferr

edox

in)

51,

21F

erra

roet

al.

(200

7)B

phC

Bur

khol

deria

xeno

vora

nsLB

400

2,3-

dihy

drox

ybip

heny

ldio

xyge

nase

32E

ltis

etal

.(1

993)

Lacc

ase

Pyc

nopo

rus

cinn

abar

inus

Mul

ticop

per

oxyd

ase

56O

tterb

ein

etal

.(2

000)

LinB

Sph

ingo

mon

aspa

ucim

obili

sU

T26

Hal

oalk

ane

deha

loge

nase

33O

akle

yet

al.

(200

4)N

bzA

(NB

DO

)C

omam

onas

sp.

JS76

5N

itrob

enze

nedi

oxyg

enas

e(o

xyge

nase

com

pone

nt)

50,

23Le

ssne

ret

al.

(200

2)N

icX

Pse

udom

onas

putid

aK

T24

402,

5-di

hydr

oxyp

yrid

ine

diox

ygen

ase

39Ji

mén

ezet

al.(

2008

)O

aGS

TO

chro

bact

rum

anth

ropi

Glu

tath

ione

-S-t

rans

fera

se22

Fav

alor

oet

al.

(199

8)

Seq

uenc

esof

allp

rote

ins

are

show

nin

Fig

.S

1.

Fig. 2. Immune response of C. dromedarius to a cocktail of 12environmentally relevant proteins.A. SDS-PAGE analysis of the source antigens. The proteins usedin the inoculation procedure (see Table 1) are shown to verify theirconcentration and purity. 0.1–1.0 mg of each of the proteins wasloaded per lane and the 12% polyacrilamide gel stained withCoomassie blue.B. Western blot of proteins fractionated in a denaturing gel andprobed with a 1/100 dilution of the camel serum after 5 weeks ofimmunization. 100 ng of protein was loaded in each lane.C. ELISA assays. 500 ng of each protein was bound to the bottomof a microtitre plate treated with a 1/100 dilution of sera comingfrom the same camel either before (pre-immune) or after theinoculation procedure. The error bars represent the results of fiveseparate experiments.

4 O. Zafra et al.


panned against each of the enzymes used as antigens,and the resulting binding pools amplified, re-packagedand subject to second panning procedure. The phagelibraries originated in the second enrichment were kept asthe bearers of the prime antigen-specific VHHs for eachprotein. We were able to isolate M13-VHH clones for allenzymes of the inoculation cocktail, except for thea-subunit of the benzylsuccinate synthase (BssA) of T.aromatica, which (as mentioned above) failed to producea significant immune response, in any case (Fig. 2).

The pool of M13-VHH clones recovered from the panningagainst the purified arsenate reductase (ArsC) of Staphy-lococcus aureus(Table 1), an enzyme that is dependenton thioredoxin for its activity (Messens and Silver, 2006)was picked for further analysis. ArsCS. aureus is functionallyidentical, but structurally unrelated to a second arsenatereductase from S. meliloti, present also in the inoculationcocktail, which runs the same reaction through an alter-native mechanism that uses instead glutaredoxin ascofactor (Yang et al., 2005). This circumstance was instru-mental to estimate the specificity characteristics of theanti-ArsCS. aureus M13- VHH pool and, by extension, those ofthe other pools as well. Figure 3 shows the reaction of theanti-ArsCS. aureus phage antibody library in a Western blotloaded with the unrelated, purified ArsCS. meliloti (lane 1),with the cognate ArsCS. aureus protein (lane 2), and with theextract of an E. coli strain (lane 3) transformed with aplasmid encoding the arsC1 gene of P. putida (Cánovaset al., 2003), which encodes a reductase of the sameprotein family as ArsCS. aureus (note that the purified ArsCproteins originate a doublet corresponding to their

reduced and oxidized forms). Figure 3 shows that theanti-ArsCS. aureus pool directly retrieved from the secondpanning procedure (see above) is not only specific for itstarget protein when facing the second ArsC, but that it cancross-react with a non-identical protein belonging to thesame functional family (Fig. S1). This suggested that thephage pool contained antibodies that target linearepitopes shared by the two related ArsC proteins (Fig.S1). The same Fig. 3 reveals virtually no cross-reactionwith any other protein of the E. coli extract (lane 3).Because the phage pool used in this experiment still con-tains a mixture of anti-ArsCS. aureus VHHs, the library thusbehaves in these experiments similarly to a polyclonalanti-serum.

To gain indications on the specificity and functionaldiversity of the antibodies present in every library weisolated 120 individual M13-VHH clones of the each of thepools from the pannings against the ArsCS. aureus andArsCS. meliloti proteins, and tested them separately in anELISA similar to that shown in Fig. 2C, excepting that inthis case a POD-conjugated anti-M13 antibody was usedto detect attachment of the viral particles to the antigens(see Experimental procedures). Each clone was thenchecked for binding the respective protein as well asagainst whole-cell extracts of either E. coli or P. putida.The results of Fig. 4 reveal some interesting details of thecorresponding antibody pools. First, most specimens ofthe libraries had considerable affinities for the cognatetarget protein and none at all for the other (not shown).This demonstrated that the panning procedure couldeffectively segregate subpopulations of VHHs generated inthe same animal for specific targets. Second, the librariescontained specimens that differed in their affinities andprobably reflected a diversity in their sequences and theirway of interacting with the matching antigen. Finally, noneof the individual M13-VHH clones presented any bindingwhatsoever to protein lysates of either E. coli (Fig. 4A) orP. putida (Fig. 4B). This meant that the procedure forgenerating the anti-ArsC pools (and for the same reason,any other protein of the cocktail) filtered any backgroundof reactivity against bacterial proteins that may have occa-sionally elicited an immune response in the camel beforeinoculation.

Quantification and significance of VHH diversity

To examine the diversity of the best-binder sequences inthe protein-specific VHH pools, 120 independent clones ofeach of the 11 separate libraries were subject to the sameELISA analysis as in Fig. 4. Those that displayed thestrongest signals (a total of 126 clones) were recoveredand the corresponding DNA sequenced for examiningintra-pool and inter-pool variability. Table 2 summarizesthe results, which can be consulted in detail in Fig. S3. As

Fig. 3. Specificity of the anti-ArsCS. aureus M13-VHH library. The gelto the left shows the appearance of the two purified ArsC proteinsfrom either S. meliloti (glutaredoxin-dependent, lane 1) or S. aureus(thioredoxin-dependent, lane 2) along with that of an extract of anE. coli strain bearing a plasmid with the cloned ArsC protein of P.putida (lane 3), which belongs to the thioredoxin-dependent class(see Fig. S2). Note that the purified ArsC proteins produce adoublet corresponding to the oxidized (top band) and the reduced(lower band) forms. The same gel is shown to the right blotted andprobed with the anti-ArsCS. aureus M13-VHH library raised after tworounds of panning against the cognate antigen. The Western blotreveals a lack of cross-reactivity of the anti-ArsCS. arureus M13-VHH

library for the ArsCS. meliloti counterpart, as well as a sensitive andspecific recognition of the reductase from P. putida, which belongsto the same protein family (only the oxidized form seems to survivethe harsh sample preparation procedure in this case).



Fig. 4. Diversity of the anti-ArsC M13-VHH

libraries. 120 phage clones were isolated fromeach of the libraries resulting from two roundsof panning against either ArsCS. meliloti (panel A)or ArsCS. aureus (panel B) and examinedseparately for binding their respective purifiedantigens or whole-cell extracts of E. coli andP. putida, as indicated. Binding was measuredas absorbance at 492 nm in an ELISA assay(see Experimental procedures).

Table 2. Diversity of VHH sequences in the pools of nanobodies against each of the inoculated proteins.

NicX ArsCSa ArsCSm BphC LinB AlkB OaGST Lacc NBDO BPDO BssA BzCoAR

Totala 14 10 9 16 10 12 16 10 14 6 0 9Differentb 11 8 7 15 1 6 6 8 6 5 0 5~ % Divc 79 80 78 94 10 50 38 80 43 83 0 56

Best binders from the libraries isolated after two rounds of panning against the proteins indicated. Note that binders classified here as differentVHH may derive from the same B cell clones if they share the same CDR3 sequence (See Fig. S3).a. Only those that produced a strong signal in an ELISA assay were picked for DNA sequencing, thereby the different number of clones ineach case.b. Diversity of DNA sequences encoding VHH with strong binding to each of the targets.c. Gross projection of VHH diversity breakdown for every inoculated protein.

6 O. Zafra et al.


a whole, we found 79 VHH variants out of the 126sequenced clones, but the distribution of diversity wasvery variable depending on the target protein. The numberof dissimilar clones ranged from only 1 strong binder inthe case of LinB (10 out of 10 sequenced were identical)to a high variability (15 different out of 16 clones sampled)in the case of the anti-BphC binders. That the pool ofVHHs elicited by proteins with little immunogenicity stillcontained strong binders (albeit with different diversity)indicated that the corresponding panning allowedamplification of excellent antibodies against otherwisepoor antigens.

Phylogenetic analysis of the sequences of the 79 differ-ent VHHs (Fig. S4) suggested also some hints on theemergence of the variants during the immunization. Obvi-ously, most of the sequences clustered according to theantigens used for their panning, as would be expectedfrom their high target specificity. Yet, while those anti-bodies selected to bind mono-component enzymes didcluster in distinct groups (for instance those againstBphC, ArsCS. aureus and ArsCS. meliloti, AlkB and OaGST), thedistribution of VHHs against multi-component enzymeswas scattered through various out-groups (e.g. NBDO,BPDO and BzCoAR). This clearly reflects that the pools ofVHHs for enzymes with multiple subunits contain antibodyspecies for more than one component, i.e. each of theout-groups may bind different components of a givenenzyme (Fig. S4). When the distribution of VHH sequenceswas compared with the phylogenetic distances betweenthe proteins of the antigenic cocktail (Fig. S5), we foundpossible correlation between the evolutionary distance ofany two antigens and the relationship between the corres-ponding antibody sequences. For instance NicX andPBDO, the sequences of which are evolutionarily close,trigger production of VHH antibodies that, despite theirspecificity for their respective proteins, map in somewhatneighbouring locations in the phylogenetic tree (Fig. S4).It is thus possible that the maturation route of the corres-ponding VHHs when the same animal is exposed simulta-neously to related proteins includes an early recognitionof antigenic features common to the proteins used forimmunization.

Regulation of the bph cluster of B. xenovorans LB400exposed with anti-BphC VHH-M13

As the most diverse pool of strong binders originated inthe panning towards BphC (see above), we surveyed theantibody library for specific clones tailored for particularpurposes. BphC is indigenous of strain B. xenovoransLB400 (Goris et al., 2004), a remarkable bacterium ableto degrade aerobically a large variety of polychlorobiphe-nyls (PCBs; Denef et al., 2004; Chain et al., 2006). Thisenzyme is an Fe2+-containing, oxygen-sensitive extradiol

dioxygenase responsible for the meta-cleavage of 2,3-dihydroxybiphenyl, the key step in the biphenyl degrada-tion pathway (Eltis et al., 1993; Hofer et al., 1993). Thegene bphC in this strain forms part of the longer bphcluster of genes (Fig. 5A) for degradation of biphenyl andco-metabolism of chlorinated derivatives (Erickson andMondello, 1992). One intriguing, barely explored aspect ofthis catabolic system is the regulation of its expression bypathway substrates and/or gratuitous inducers (Parnellet al., 2009). The literature reports that the activity of theupstream promoters of the operon is semi-constitutive inthat there is a considerable basal level of expression inthe absence of any inducer along with a degree of induc-tion in the presence of the pathway substrate, biphenyl(Beltrametti et al., 2001). On the other hand, the leadingenzyme of the pathway, the multi-component biphenyldioxygenase encoded by the bphA segment of the operon(Fig. 5A) is very similar to many other ring-dioxygenases(Pérez-Pantoja et al., 2009) and it is probably quitepromiscuous in its substrate range (Furukawa, 2000;Furukawa et al., 2004). If the BphA enzyme originated ina gene cluster formerly associated to another degradativeroute, it is then possible that its expression is still regu-lated by the former inducers (Pieper and Seeger, 2008).The availability of anti-BphC antibodies allowed a freshlook to these two questions in the native strain B. xeno-vorans LB400, rather than relying on the use of heterolo-gous reporter systems inserted in unknown sites of thechromosome (Beltrametti et al., 2001) and thus prone toartefacts. Figure 5 shows the result of using BphC levelsas a proxy of the expression of the entire bph pathwaywhen B. xenovorans LB400 is grown under various con-ditions. To this end, we used one singular VHH-M13 cloneout of the anti-BphC library that was particularly efficientat recognizing the denatured protein in a Western blot.Lane 4 of Fig. 4B shows that expression of BphC wasbarely detectable in the absence of any aromatic inducer.On the contrary, exposure of the culture to biphenylvapours (lane 6) boosted expression of this protein by> 100 fold. Unexpectedly, comparable levels of theenzyme were reached when non-pathway substratessuch as naphthalene or toluene were used as alternativeinducers, and to a much lesser extent, with benzene.These results document not only the lack of expression ofthe bph genes when cells grown on organic acids suchas citrate, but also their strong responsiveness to thepresence of BphA substrates-to-be other than biphenyl,the nominal target of the pathway. That responses toapparently gratuitous effectors are so strong rather thanmarginal (B. xenovorans LB400 does not grow on naph-thalene, toluene or benzene; Denef et al., 2004; Pieperand Seeger, 2008), suggests that the promiscuity of theregulatory system might be ultimately beneficial in sitespolluted with multiple hydrocarbons and colonized by



various species (Martinez-Perez et al., 2007; de Lorenzoet al., 2010), an issue that is beyond the scope of thisarticle. In any case, the results of Fig. 5 highlight the valueof directly detecting pathway products rather than usingreporter genes for addressing the problem of regulatoryactivity and specificity in environmental bacteria (deLorenzo and Perez-Martin, 1996).

In vivo targeting of BphC activity

Given the high diversity of the anti-BphC VHH-M13 pool wereasoned that some molecular specimens of the antibo-dies could interact intracellularly with the protein surfacesof the dioxygenase in vivo, as this could make post-translational control of its enzymatic activity feasible. Tostudy this possibility we recloned in masse the wholeof anti-BphC VHH sequences from its original vector(pXylE5EHis, see Experimental procedures) into theframe of the intracellular expression plasmid pTB7(Jurado et al., 2006). This vector adds a thioredoxinmoiety to the N-terminus of the VHH sequence, what isknown to facilitate the stable folding of recombinant anti-bodies in an active form in the cytoplasm of bacteria(Jurado et al., 2006). The pool of pBT7-derived plasmidswas then transformed into an E. coli strain carrying thebphC+ plasmid pCKBphC. The presence and activity ofBphC can be easily monitored because of its 2,3-dioxygenase activity on plain catechol. This results inproduction of a characteristic yellow colour (a semialde-

hyde that can be followed spectrophotometrically) whencells are exposed to such a surrogate substrate. Follow-ing transformation with the whole VHH pBT-7-based library,E. coli cells were plated on selective medium to ensureretention of pCKBphC (Cm) and the intracellular expres-sion vector (Ap). Colonies were then let to develop andsubsequently sprayed with catechol. Although the VHH hadbeen enriched for binding BphC in the panning proceduredescribed above, most of the resulting E. coli coloniesturned yellow, suggesting that the corresponding antibo-dies were not expressed, not folded in an active form, notrecognizing the BphC epitopes in their native conforma-tion, or not targeting sites important for the dioxygenaseactivity. However, we also found the presence of a 0.1% ofthe colonies that did not turn yellow when subject to thesame treatment. As a control, 100% of the transformantsof the same bphC+ E. coli host with an unrelated VHH

library gave yellow colonies when spread with catechol(not shown).

Two of the colourless colonies containing anti-BphC VHH

clones (designated F and R) were cultured in liquidmedium and their catechol 2,3 dioxygenase activity mea-sured in cells at various growth stages. As shown inFig. 6A and B the results confirmed the seminal observa-tion that BphC was intracellularly inhibited by the corres-ponding antibodies. One possibility was that suchintracellular VHHs could have targeted a site in the proteinthat was important for its enzymatic activity, whetherbecause interfering with inter-monomer interactions (the

Fig. 5. Regulation of the bph operon of B. xenovorans exposed through the expression profile of BphC.A. Organization of the bph gene cluster. Degradation of biphenyl and PCBs in this strain is determined by multi-component dioxygenase(encoded by leading genes bphA1-bphA4) which is co-transcribed along with an array of catabolic genes bphB–bphD, which includes that forthe meta enzyme (BphC) that cleaves 2,3 dihydroxybiphenyl to yield a coloured hemialdehyde (Pieper and Seeger, 2008). The whole operonis transcribed from a promoter region upstream of bphA1, which is regulated by a transcriptional factor encoded by bphR (Beltrametti et al.,2001).B. Western-blot assay of different BphC-containing samples. Lanes 1–3 were loaded with controls: pure protein, E. coli transformed with abphC + plasmid and its corresponding insert-less vector counterpart, as indicated. The other lanes contain crude extracts of B. xenovoransLB400 growing on citrate (0.2%) as the sole carbon source and exposed to saturating vapours of benzene, biphenyl, naphthalene andtoluene. The blot was probed with a 1/2000 dilution of one of the clones of the anti-BphC M13-VHH library selected for optimal binding to linearepitopes.C. Quantification of BphC contents of cells. The numbers of the X-axis correspond to the densitometry of the protein signals of the lanes ofthe Western blot before. Bars reflect the results of � 3 independent replicates.

8 O. Zafra et al.


enzyme is an octamer) or by blocking the active centre(Lauwereys et al., 1998). To gain some insight into thisquestion we sequenced both inhibitory VHH clones F and Ras well as two non-inhibitory binders (clones M and X).Comparison of the resulting sequences (Fig. S6) andhomology modelling of each of them (Fig. 6C) pinpointeda short region around amino acid position 30 (placed in

the H1 loop), which appeared to discriminate those thatsimply bound BphC from those which also inhibited itsactivity (Fig. 6C). When such position 30 was occupied bya proline, the structural prediction for the surroundingresidues of the antibody was that of a non-structured site.In contrast, when the same site held a leucine or a threo-nine, the tridimensional modelling anticipated the forma-tion of a short a-helix. The few examples of crystals of VHH

domains bound to an enzyme (e.g. lysozyme; De Genstet al., 2006b) show most inhibitory variants to diversify inthe H3 loop (De Genst et al., 2006b). In contrast, the H3region is largely conserved in nanobodies against BphC,inhibitory or not, isolated by panning (Fig. S6). We specu-late that such H3 region accounts in most cases forstrongly binding a permissive surface of the target proteinbut close to a site required for activity, whereas changes inthe H1 loop determine the inhibitory ability if the VHH.Alternatively, the divergence between inhibitory and non-inhibitory VHHs may simply stem from their different affinityfor the same target. These hypotheses are currently underexamination.

Conclusion

VHH domain sequences reconstructed from the RNA oflymphocytes of camels inoculated with biodegradativeenzymes facilitate the investigation of intricate questionson the biology of environmental bacteria. As shownabove, a large structural and functional diversity of suchantibodies can be generated from a single shot of ananimal with a mixture of many proteins, and the corres-ponding VHHs expressed in unlimited amounts in aprokaryotic system for specific applications. It is remark-able that, despite the very different early immuneresponse to such an antigenic cocktail (Fig. 2), we wereable to collect large VHHs libraries against 11 out of the12 proteins included in the inoculation protocol (in factmore, if we count multi-component enzymes). The libra-ries specific for each protein behaved as de facto poly-clonal sera, as they recognized both linear epitopes in adenaturing fractionation system and afforded the recog-nition of non-identical proteins belonging to the samefamily. We have illustrated these properties above onlyfor the ArsC reductase of S. aureus and the BphC dioxy-genase of B. xenovorans. But provided enough diversityof VHHs, the conclusions are likely to be true for theother proteins as well. Such serum-like preparationscontaining multiple types of VHHs against the sameprotein and cross-reaction with similar ones have anextraordinary potential as an environmental diagnosetool for monitoring the presence of distinct biodegrada-tive enzymes in raw metaproteomes (Benndorf et al.,2007; Schneider and Riedel, 2009), a development thatis at present pursued in our Laboratory. Yet, the most

Fig. 6. Intracellular inhibition of catechol 2,3 dioxygenase in vivowith anti-BphC nanobodies.A. C2,3O of E. coli CC118 cells co-transformed with plasmidpCKBphC and each of the pTB7 vector derivatives expressing theVHH clones indicated. Culture samples were taken at the growthstages indicated. Activity is expressed as % of the C2,3Omeasured in a culture of cells transformed with pCKBphC only.Note the high activity of the controls with a VHH variant againstanother protein of the inoculation cocktail (NicX), or thoseexpressing VHHs that bind BphC in an ELISA test but not bringingabout any C2,3O inhibition when expressed in the cytoplasm (VHH

clones X and M). In contrast, VHHs named F and R caused a strongquenching of the enzyme.B. Qualitative BphC inhibition assays. 1 ml of bacterial cultures ofE. coli strains transformed with pCKBphC and the pTB7 derivativesencoding either the inhibitory VHH clone F or the non-inhibitory VHH

clone X were added with 15 ml catechol 1% and the colour let todevelop for 1 min.C. Predicted tridimensional structures of VHH that bind BphC usedfor modelling. The arrangement of residues in the neighbourhoodof amino acid position 30 of two inhibitory (F, R) and twonon-inhibitory (M,X) nanobodies are shown. The model uses theknown structures of a variable fragment of the humanized antibodyC25 (pdb : 2gcy) and a camel VHH domain (pdb : 1mvf) as threadingtemplates. Note the structuring of the region in the shape of a shorta-helix with a protruding amino acid side-chain in the case of theinhibitory nanobodies. The same region is absent in thenon-inhibitory counterparts.



important value of this approach is the possibility toselect specific clones out of the VHHs pools that areoptimal for particular uses. For instance, we have shownthat the same starting anti-BphC VHH pool contains sin-gular nanobody specimens that detect the denaturedprotein with a high specificity and sensitivity in a Westernassay (Fig. 5). The same pool includes VHH variants alsothat interact (and eventually block) the same structuredprotein in an in vivo cellular context. One can easily envi-sion the large number of biological and biotechnologicalissues that having such tools in hand may allow tackling.Only two of them have been outlined in this work butare currently the subject of ongoing studies. First, theproblem of regulatory promiscuity in pathways recentlyevolved for degradation of xenobiotic compounds (deLorenzo and Perez-Martin, 1996) a subject that canhardly be addressed exclusively with genetic means andreporter technology. Second, the possibility of usingnanobodies as intracellular enzyme inhibitors able toknock out distinct pathways at a post-translational leveland thus create reversible metabolic phenotypes. Webelieve that the use of nanobodies of the sort presentedhere will be helpful in the eventual outcome of thesestanding questions.

Experimental procedures

Camel immunization and VHH library construction

The immunogenic mixture used in the inoculation procedurewas prepared as follows. A protein cocktail containing 1 mgof each of the samples listed in Table 1 was adjusted to avolume of 12.5 ml in buffer 100 mM Tris-HCl pH 7.5, 50 mMNaCl. At the moment of inoculation, 2.5 ml of this mixturewas combined with 2.5 ml of Gerbu Veterinary VaccineAdyuvant (http://www.gerbu.de) and the resulting ~5 ml anti-genic pool injected subcutaneously into one young (4 year)male dromedary (C. dromedarius) each time along a 5 weekperiod (Saerens et al., 2004). One week after the last shot,50 ml of blood was extracted and mixed with 50 ml of theleukocytes culture medium RPMI-1640 (Sigma). The periph-eral lymphocytes present in the blood sample were isolatedby centrifugation in a sterile density gradient using Ficoll-Paque PLUS (http://www.gelifesciences.com). Purified lym-phocytes were then disrupted using TRIzol reagent (http://www.invitrogen.com) and their RNA precipitated with 1volume of isopropanol with NaAc 0.3 M pH 5.0 at -70°C andwashed with 70% cold EtOH. Poly-A tailed mRNA was recov-ered using the Oligotex mRNA Purification System (http://www.qiagen.com). Finally, 2.5–5 mg of the thereby purifiedmRNA was used as a template in a retrotranscription reac-tion (iScript cDNA synthesis kit, http://www.bio-rad.com).This originated the primary pool of cDNA sequences encod-ing camel immunoglobulins. In order to amplify the 5′ seg-ments of such sequences that match the VHH domains of thecorresponding antibodies, 0.2 mg of the cDNA pool wasamplified with primers CALL001 and CALL002 that anneal,respectively, the leader sequence and the CH2 exon of the

cognate mRNA (Saerens et al., 2004). Although fragments ofdifferent size were generated as products of the PCR reac-tion, only those ~600 bp can encode the variable domains ofthe corresponding heavy-chain-only antibodies. DNA bandswithin that size range were purified and used as a templatein a second PCR with oligonucleotides VHHSfiI (5′-GTCCTCGCAACTGCGGCCCAGCCGG CCATGGCCCAGGTGCAGCTGGTGGA-3′) and VHHNotI (5′-GGACTAGTGCGGCCGCTGAGGAGAC GGTGACCTGGGT-3′). These primersgenerated 450 bp DNA fragments flanked by unique SfiI andNotI restriction sites that enable the encoded sequence to befused in frame to the pIII apical protein of M13 phage bycloning in the corresponding SfiI–NotI sites of vectorpXylE5EHis (see pedigree below). Ligations were trans-formed into cells the F+ E. coli TG1 strain and were selectedin LB-agar plates supplemented with ampicillin (150 mg ml-1)and glucose (2%), originating ~106 independent clones withonly 1% of religated vector background. A M13 phage libraryfor packaging of the DNA sequences and display of theresultant VHH domains was produced by infecting the pool ofE. coli TG1 transformants with the interference-resistanthelper phage VCS-M13 (http://www.agilent.com) following astandard protocol (Clackson and Lowman, 2004). Phageswere recovered from culture supernatants by precipitationwith PEG8000 and NaCl, and the sediment resuspendedand kept in TE buffer. This method produced a VHH-M13library with a titre ~109 of PFUs embodying ¥1000 represen-tation of the primary diversity of the VHH pool. Phages werestored at -80°C for further use with no apparent decrease ofthe titre over time.

Biopanning of antigen-binding phages and enrichmentfor strong binders

Microtitre plates for panning the M13-VHH library were pre-pared as follows. Diluted solutions (10 mg ml-1) of each of theproteins used for the inoculation of the camel (Table 1) wereset in 8 mM Na2HPO4, 1.5 mM KH2PO2, 3 mM KCl, 137 mMNaCl pH 7.0 (PBS) and separately adsorbed to 94-well Max-isorb immunoplates (http://www.nuncbrand.com) by adding50 ml to each well and incubating during 12 h at 4°C. Thewells were then emptied and the absorbed protein blocked byadding 200 ml of a solution of PBS, with 0.1% Tween-20, 3%skim milk and 1% and BSA. After 2 h, the blocking mix wasreplaced by 2 ¥ 1011 PFUs of the VHH library packaged in M13capsids (see above) suspended in the same solution. Thesewere let to react for 1 h at room temperature, and unboundphages removed by washing 20 times for 1 min with200 ml well-1 of PBS added with 0.1% Tween-20. Binderswere eluted from the plate after 5 min incubation with50 ml well-1 with 0.1 M glycine, pH 5.0. Thereby released VHH-M13 particles were recovered from the wells and neutralizedwith 50 ml of 1.0 M Tris HCl, pH 7.5. The resulting suspensionwas directly used for infecting E. coli XL-1 Blue cells and ApR

clones counted for an estimation of diversity and efficacy ofthe procedure. As this method favours the capture of strongbinders, the pool of clones was re-packaged in M13 capsidesby infection with helper phage VCS-M13 (see above), theviral pool re-isolated as before and, where indicated,re-panned for enrichment of VHHs with a growing affinity forthe target.

10 O. Zafra et al.


ELISA

Antigens under study (whether purified proteins or whole cellextracts) were bound to the ELISA plates as explained above.To examine the overall response of camels following inocu-lation with the cocktail of proteins, sera from the same animalbefore and after exposure to the antigens were diluted ¥ 100in PBS buffer, 50 ml deposited in the corresponding welland let to react for 1 h with the bound proteins. Plates werethen washed with PBS and bound IgG-type antibodiesrevealed with Protein A conjugated with peroxidase usingo-phenylenediamine (Sigma) as the reaction substrate. Thecolour was let to develop in the darkness for 10 min and thenstopped with 20 ml well-1 of 3 M HCl, following which theabsorbance of the samples at 492 nm was determined. Fordetection of binding of M13-VHH specimens to the proteinsimmobilized in the plates, the phages were applied to thewells as explained above and subjected to three washeswith 100 ml of PBS. Detection of those phages attachedto the antigens was made by applying 50 ml of a PBS solutionto each well supplemented with 3% skim milk and bearinga 1/5000 dilution of the HRP/anti-M13 monoclonal anti-body conjugated with peroxidase (Amersham PharmaciaBiotech). After four washing steps with 100 ml of PBS, thepresence of anti-M13 antibodies was exposed by addingo-phenylenediamine to the plates, as before.

Bacterial strains, culture conditions andsample preparation

Strain E. coli DH5a (F- endA1 glnV44 thi-1 recA1 relA1gyrA96 deoR nupGf80dlacZDM15 D[lacZYA-argF] lacU169,hsdR17, l-; Sambrook and Russell, 2001) was routinely usedfor cloning purposes. E. coli TG1 (F’ [traD36 lacIq DlacZM15proAB+] supE DhsdM-mcrB thi Dlac-proAB; Sambrook andRussell, 2001) was used for library construction, while E. coliXL-1 Blue (endA1 gyrA96 thi-1 recA1 relA1 lac glnV44 F’[Tn10 proA+ lacIq DlacZM15] hsdR17; Sambrook and Russell,2001) was preferred for phagemid amplification and rescue.E. coli CC118 (phoA20 thi-1 rspE rpoB argEAm recA1 (Manoiland Beckwith, 1985) was used for expression of the ars1operon of P. putida KT2442 and the bphC gene of Bulkhold-eria xenovorans LB400 (see below). The same E. coli strainwas used in the experiments for testing intracellular inhibitionof the bphC-encoded enzyme, 2-hydroxybiphenyl dioxyge-nase. All E. coli strains used in this work were grown inLB media (Miller, 1992) supplemented with ampicillin(150 mg ml-1), kanamycin (50 mg ml-1) or chloramphenicol(30 mg ml-1) when needed. P. putida KT2440 (Franklin et al.,1981) was cultured also in LB supplemented with the amend-ments indicated in each case. Burkholderia xenovoransLB400 (Goris et al., 2004) was grown in minimal medium M9added with 0.2% citrate. Cells cultured in these conditionswere exposed, where specified, to saturating vapours ofbenzene, biphenyl, naphthalene and toluene. Bacterial cul-tures were in all cases incubated at 30°C with shaking(170 rpm) until an A600 = 0.8 was reached. 1 ml samples wasthen collected by centrifugation (14 000 r.p.m., 5 min) anddirectly resuspended in 100 ml of protein loading buffer. Fol-lowing a short sonication, samples were denatured 10 min at100°C and submitted to protein analysis as explained below.

Plasmid constructs

DNA was manipulated using standard methods (Ausubelet al., 1994). pXylE5EHis (ApR) is a derivative of plasmidp6AC3g3 (Fernandez et al., 2000) used to express single-chain antibodies by phage-display, which allows yellow/whitescreening as a result of the presence of a catechol 2,3-dioxygenase encoding gene (xylE) in its polylinker cloned asa SfiI/NotI fragment. pVCl-1 contains the arsenic resistancecassette ars1 from P. putida KT2440 (Cánovas et al., 2003).For its construction, the corresponding ars1 operon wasamplified from genomic DNA by means of oligonucleotidesFWDArs1 (5′-CGGCAAGCTT GAGCGTATCCAGGC-3′) andRVSArs1 (5′-CGTCCCGGAATTCGAGGCGATTG-3′). Theamplified fragment (3.1 kb) was then cloned in KmR vectorpVLT33 (de Lorenzo et al., 1993) as an EcoRI–HindIIIsegment. For construction of pCKBphC (CmR), the PstI frag-ment from pDD5301 (Dowling et al., 1993) containing bphC(encoding 2,3 OH biphenyl dioxygenase) was cloned into thelow copy vector pCK01 (Fernández et al., 1995). The pTC-VHH (ApR) plasmid series harbouring VHH domain sequencesexpressed as protein fusions to thioredoxin were generatedafter purifying the corresponding NcoI/NotI fragments of theVHH pool first captured in vector pXylE5EHis (see above) andre-cloning them into the same sites of the intracellular expres-sion vector pTB7 (Jurado et al., 2006).

Protein electrophoresis and immunoblotting

Denaturing SDS-PAGE was performed in 4% stacking and10–12% separating gels (acrylamide : bisacrylamide 29:1;Bio-Rad), using the MiniProtean electrophoresis system (Bio-Rad) following standard protocols (Ausubel et al., 1994;Fraile et al., 2001). For immunoblotting, the proteins weretransferred to polyvinylidene difluoride membranes (Immo-bilon, Millipore) using a semi-dry electrophoresis transferapparatus (Bio-Rad) and then blocked in PBS buffer contain-ing 3% skimmed milk and 0.1% Tween-20 for 1 h. Whereindicated, the blots were subsequently incubated at roomtemperature in the same buffer (without detergent) with a1/100 dilution of the inoculated camel serum for 1–2 h. Themembranes were then rinsed with PBS, incubated withprotein A conjugated with peroxidase and the proteins bandsrevealed with the BM Chemoluminiscence Blotting Substrate-POD (horseradish peroxidase) kit (Roche). After a 1 min inthe dark, the blots were exposed to an X-ray film (X-OMAT,Kodak). Alternatively, the pools of M13 particles apically pre-senting VHH domains were used instead of serum to theblotted membranes for detection of target proteins. To thisend, membranes were placed in phage suspensions diluted(typically 1/2000) in a PBS buffer supplemented with 3%skimmed milk, 0.1% Tween-20 and 0.1% sodium deoxycho-late, followed by a 45 min incubation at room temperaturewith mild shaking. Unbound phages were washed out withfour 5 min rinses with the same PBS/Tween-20/sodiumdeoxycholate buffer but devoid of milk. After washing withPBS, detection of M13 capsides bound to the blotted proteinswas made by immersion of the membrane in a 1/5000 dilutionof HRP/anti-M13 Monoclonal Conjugate and revealed withthe chemoluminiscence procedure mentioned before. Whereneeded, signals in the membranes were integrated using the



Quantity One Software package included in a VersadocImager System (Bio-Rad).

Enzymatic assays

2,3-dihydroxybiphenyl-2,3-dioxygenase activity was esti-mated using plain catechol as a substitute of the authenticenzyme substrate (2,3 dihydroxybiphenyl). Bacterial cultureswere grown in LB medium and the catechol 2,3-dioxygenasereaction triggered at the points indicated by adding 7.5 ml of1% catechol to 1 ml of culture of the tested strains. After1 min of incubation at room temperature, cells were centri-fuged 2 min at 14 000 r.p.m. and the absorbance of thesupernatant at 375 nm measured. Concentration of the semi-aldehyde resulting from the reaction was estimated using anextinction coefficient (e) of 46 000 M cm-1 (Velázquez et al.,2006).

Sequence analysis and protein modelling

Amino acid sequences were aligned and phylogenetic treesgenerated using the CLUSTALW algorithm available atthe EMBL-EBI server (http://www.ebi.ac.uk/Tools/clustalw;Chenna et al., 2003). Protein modelling was done in the3D-JIGSAW (Bates et al., 2001) and ESyPred3D (Lambertet al., 2002) sites using pdb’s 2gcy and 1mvf as templates.Protein structures were represented using PYMOL molecularvisualization system (http://www.pymol.org).

Acknowledgements

This work would have been impossible without the generousgift of purified proteins from each of the Laboratories whosereferences are cited in Table 1. Authors are indebted to L. A.Fernández for his help to set up the camel antibody platformand his continuous support. S. Muyldermans is acknow-ledged also for important hints on VHH technologies. Thisresearch was funded by grants of the CONSOLIDERprogram of the Spanish Ministry of Science and Innovation,by EU Grants BACSINE and MICROME and by Funds of theAutonomous Community of Madrid.

References

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D.,Seidman, J.G., Smith, J.A., and Struhl, K. (1994) CurrentProtocols in Molecular Biology. New York, NY, USA: JohnWiley & Sons.

Bates, P.A., Kelley, L.A., MacCallum, R.M., and Sternberg,M.J. (2001) Enhancement of protein modeling by humanintervention in applying the automatic programs3D-JIGSAW and 3D-PSSM. Proteins 45 (Suppl. 5): 39–46.

van Beilen, J.B., Holtackers, R., Luscher, D., Bauer, U.,Witholt, B., and Duetz, W.A. (2005) Biocatalytic productionof perillyl alcohol from limonene by using a novel Myco-bacterium sp. cytochrome P450 alkane hydroxylaseexpressed in Pseudomonas putida. Appl Environ Microbiol71: 1737–1744.

Belkin, S. (2003) Microbial whole-cell sensing systemsof environmental pollutants. Curr Opin Microbiol 6: 206–212.

Beltrametti, F., Reniero, D., Backhaus, S., and Hofer, B.(2001) Analysis of transcription of the bph locus ofBurkholderia sp. strain LB400 and evidence that the ORF0gene product acts as a regulator of the bphA1 promoter.Microbiology 147: 2169–2182.

Benndorf, D., Balcke, G.U., Harms, H., and von Bergen, M.(2007) Functional metaproteome analysis of proteinextracts from contaminated soil and groundwater. ISME J1: 224–234.

Bombach, P., Richnow, H.H., Kastner, M., and Fischer,A. (2010) Current approaches for the assessment ofin situ biodegradation. Appl Microbiol Biotechnol 86: 839–852.

Breese, K., Boll, M., Alt-Mörbe, J., Schägger, H., and Fuchs,G. (1998) Genes coding for the benzoyl-CoA pathway ofanaerobic aromatic metabolism in the bacterium Thaueraaromatica. Eur J Biochem 256: 148–154.

Cánovas, D., Cases, I., and de Lorenzo, V. (2003) Heavymetal tolerance and metal homeostasis in Pseudomonasputida as revealed by complete genome analysis. EnvironMicrobiol 5: 1242–1256.

Chain, P.S., Denef, V.J., Konstantinidis, K.T., Vergez, L.M.,Agullo, L., Reyes, V.L., et al. (2006) Burkholderia xeno-vorans LB400 harbors a multi-replicon, 9.73-Mbp genomeshaped for versatility. Proc Natl Acad Sci USA 103: 15280–15287.

Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J.,Higgins, D.G., and Thompson, J.D. (2003) Multiplesequence alignment with the Clustal series of programs.Nucleic Acids Res 31: 3497–3500.

Clackson, T., and Lowman, H.B. (eds) (2004) Phage Display.A Practical Approach. New York, NY, USA: Oxford Univer-sity Press.

De Genst, E., Saerens, D., Muyldermans, S., and Conrath, K.(2006a) Antibody repertoire development in camelids. DevComp Immunol 30: 187–198.

De Genst, E., Silence, K., Decanniere, K., Conrath, K., Loris,R., Kinne, J., et al. (2006b) Molecular basis for the prefer-ential cleft recognition by dromedary heavy-chain antibod-ies. Proc Natl Acad Sci USA 103: 4586–4591.

Denef, V.J., Park, J., Tsoi, T.V., Rouillard, J.M., Zhang, H.,Wibbenmeyer, J.A., et al. (2004) Biphenyl and benzoatemetabolism in a genomic context: outlining genome-widemetabolic networks in Burkholderia xenovorans LB400.Appl Environ Microbiol 70: 4961–4970.

Dowling, D.N., Pipke, R., and Dwyer, D.F. (1993) A DNAmodule encoding bph genes for the degradation of poly-chlorinated biphenyls (PCBs). FEMS Microbiol Lett 113:149–154.

Eltis, L.D., Hofmann, B., Hecht, H.J., Lünsdorf, H., andTimmis, K.N. (1993) Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem 268:2727–2732.

Erickson, B.D., and Mondello, F.J. (1992) Nucleotidesequencing and transcriptional mapping of the genesencoding biphenyl dioxygenase, a multicomponentpolychlorinated-biphenyl-degrading enzyme in Pseudomo-nas strain LB400. J Bacteriol 174: 2903–2912.

12 O. Zafra et al.


Favaloro, B., Tamburro, A., Angelucci, S., Luca, A.D., Melino,S., di Ilio, C., and Rotilio, D. (1998) Molecular cloning,expression and site-directed mutagenesis of glutathioneS-transferase from Ochrobactrum anthropi. Biochem J335: 573–579.

Fernandez, L.A., Sola, I., Enjuanes, L., and de Lorenzo, V.(2000) Specific secretion of active single-chain Fv antibod-ies into the supernatants of Escherichia coli cultures by useof the hemolysin system. Appl Environ Microbiol 66: 5024–5029.

Fernández, S., de Lorenzo, V., and Pérez-Martín, J. (1995)Activation of the transcriptional regulator XylR ofPseudomonas putida by release of repression betweenfunctional domains. Mol Microbiol 16: 205–213.

Ferraro, D., Brown, E., Yu, C.-L., Parales, R., Gibson, D., andRamaswamy, S. (2007) Structural investigations of theferredoxin and terminal oxygenase components of thebiphenyl 2,3-dioxygenase from Sphingobium yanoikuyaeB1. BMC Struct Biol 7: 10.

Fraile, S., Roncal, F., Fernandez, L.A., and de Lorenzo, V.(2001) Monitoring intracellular levels of XylR in Pseu-domonas putida with a single-chain antibody specificfor aromatic-responsive enhancer-binding proteins.J Bacteriol 183: 5571–5579.

Franklin, F.C., Bagdasarian, M., Bagdasarian, M.M., andTimmis, K.N. (1981) Molecular and functional analysis ofthe TOL plasmid pWWO from Pseudomonas putida andcloning of genes for the entire regulated aromatic ring metacleavage pathway. Proc Natl Acad Sci USA 78: 7458–7462.

Furukawa, K. (2000) Engineering dioxygenases for efficientdegradation of environmental pollutants. Curr Opin Bio-technol 11: 244–249.

Furukawa, K., Suenaga, H., and Goto, M. (2004) Biphenyldioxygenases: functional versatilities and directed evolu-tion. J Bacteriol 186: 5189–5196.

Gomez, M.J., Pazos, F., Guijarro, F.J., de Lorenzo, V., andValencia, A. (2007) The environmental fate of organic pol-lutants through the global microbial metabolism. Mol SystBiol 3: 114.

Goris, J., De Vos, P., Caballero-Mellado, J., Park, J., Falsen,E., Quensen, J.F. III, et al. (2004) Classification of thebiphenyl- and polychlorinated biphenyl-degrading strainLB400T and relatives as Burkholderia xenovorans sp. Int JSyst Evol Microbiol 54: 1677–1681.

Hamers-Casterman, C., Atarhouch, T., Muyldermans, S.,Robinson, G., Hamers, C., Songa, E.B., et al. (1993) Natu-rally occurring antibodies devoid of light chains. Nature363: 446–448.

Harmsen, M.M., and De Haard, H.J. (2007) Properties,production, and applications of camelid single-domainantibody fragments. Appl Microbiol Biotechnol 77: 13–22.

He, Z., Deng, Y., Van Nostrand, J.D., Tu, Q., Xu, M., Hemme,C.L., et al. (2010) GeoChip 3.0 as a high-throughput toolfor analyzing microbial community composition, structureand functional activity. ISME J 4: 1167–1179.

Hofer, B., Eltis, L.D., Dowling, D.N., and Timmis, K.N. (1993)Genetic analysis of a Pseudomonas locus encoding apathway for biphenyl/polychlorinated biphenyl degradation.Gene 130: 47–55.

Jiménez, J.I., Canales, A., Jiménez-Barbero, J., Ginalski,K., Rychlewski, L., García, J.L., and Díaz, E. (2008)Deciphering the genetic determinants for aerobic nicotinicacid degradation: the nic cluster from Pseudomonasputida KT2440. Proc Natl Acad Sci USA 105: 11329–11334.

Jurado, P., Fernández, L., and de Lorenzo, V. (2006) In vivodrafting of single-chain antibodies for regulatory duty onthe s54-promoter Pu of the TOL plasmid. Mol Microbiol 60:1218–1227.

Kolok, A.S., and Sellin, M.K. (2008) The environmentalimpact of growth-promoting compounds employed by theUnited States beef cattle industry: history, current knowl-edge, and future directions. Rev Environ Contam Toxicol195: 1–30.

Kümmerer, K. (2009a) Antibiotics in the aquatic environment– a review – part I. Chemosphere 75: 417–434.

Kümmerer, K. (2009b) Antibiotics in the aquatic environment– a review – part II. Chemosphere 75: 435–441.

Lambert, C., Leonard, N., De Bolle, X., and Depiereux, E.(2002) ESyPred3D: prediction of proteins 3D structures.Bioinformatics 18: 1250–1256.

Lauwereys, M., Arbabi Ghahroudi, M., Desmyter, A., Kinne,J., Holzer, W., De Genst, E., et al. (1998) Potent enzymeinhibitors derived from dromedary heavy-chain antibodies.EMBO J 17: 3512–3520.

Leitgib, L., Kálmán, J., and Gruiz, K. (2007) Comparison ofbioassays by testing whole soil and their water extract fromcontaminated sites. Chemosphere 66: 428–434.

Lessner, D.J., Johnson, G.R., Parales, R.E., Spain, J.C., andGibson, D.T. (2002) Molecular Characterization and sub-strate specificity of nitrobenzene dioxygenase from Coma-monas sp. strain JS765. Appl Environ Microbiol 68: 634–641.

Leuthner, B., and Heider, J. (1998) A two-component systeminvolved in regulation of anaerobic toluene metabolism inThauera aromatica. FEMS Microbiol Lett 166: 35–41.

Liu, Z.H., Kanjo, Y., and Mizutani, S. (2009) Urinary excretionrates of natural estrogens and androgens from humans,and their occurrence and fate in the environment: a review.Sci Total Environ 407: 4975–4985.

de Lorenzo, V. (2008) Systems biology approaches to biore-mediation. Curr Opin Biotechnol 19: 579–589.

de Lorenzo, V., and Perez-Martin, J. (1996) Regulatory noisein prokaryotic promoters: how bacteria learn to respond tonovel environmental signals. Mol Microbiol 19: 1177–1184.

de Lorenzo, V., Eltis, L., Kessler, B., and Timmis, K.N. (1993)Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phe-notypes. Gene 123: 17–24.

de Lorenzo, V., Silva-Rocha, R., Carbajosa, G., Galvão, T.G.,and Cases, I. (2010) Sensing xenobiotic compounds:lessons from bacteria that face pollutants in the environ-ment. In Sensory Mechanisms in Bacteria: MolecularAspects of Signal Recognition. Dixon, S.S.R. (ed.). Norfolk,UK: Caister Academic Press, pp. 83–108.

Manoil, C., and Beckwith, J. (1985) TnphoA: a transposonprobe for protein export signals. Proc Natl Acad Sci USA82: 8129–8133.

Martinez-Perez, O., Lopez-Sanchez, A., Reyes-Ramirez, F.,



Floriano, B., and Santero, E. (2007) Integrated re-sponse to inducers by communication between a cata-bolic pathway and Its regulatory system. J Bacteriol 189:3768–3775.

van der Meer, J.R., and Belkin, S. (2010) Where microbiologymeets microengineering: design and applications ofreporter bacteria. Nat Rev Microbiol 8: 511–522.

Messens, J., and Silver, S. (2006) Arsenate reduction: thiolcascade chemistry with convergent evolution. J Mol Biol362: 1–17.

Messens, J., Hayburn, G., Desmyter, A., Laus, G., and Wyns,L. (1999) The Essential catalytic redox couple in arsenatereductase from Staphylococcus aureus. Biochemistry 38:16857–16865.

Miller, J.H. (1992) A Short Course in Bacterial Genetics. ColdSpring Harbor, NY, USA: Cold Spring Harbor LaboratoryPress.

Oakley, A.J., Klvana, M., Otyepka, M., Nagata, Y., Wilce,M.C.J., and Damborsky, J. (2004) Crystal Structure ofhaloalkane dehalogenase LinB from Sphingomonas pauci-mobilis UT26 at 0.95 Å resolution: dynamics of catalyticresidues. Biochemistry 43: 870–878.

Omidfar, K., Rasaee, M.J., Modjtahedi, H., Forouzandeh, M.,Taghikhani, M., Bakhtiari, A., et al. (2004) Production andcharacterization of a new antibody specific for the mutantEGF receptor, EGFRvIII, in Camelus bactrianus. TumourBiol 25: 179–187.

Otterbein, L., Record, E., Longhi, S., Asther, M., and Moukha,S. (2000) Molecular cloning of the cDNA encoding laccasefrom Pycnoporus cinnabarinus I-937 and expression inPichia pastoris. Eur J Biochem 267: 1619–1625.

Parales, R.E., Bruce, N.C., Schmid, A., and Wackett, L.P.(2002) Biodegradation, biotransformation, and biocataly-sis. Appl Environ Microbiol 68: 4699–4709.

Parnell, J.J., Denef, V.J., Park, J., Tsoi, T., and Tiedje, J.M.(2009) Environmentally relevant parameters affecting PCBdegradation: carbon source- and growth phase-mitigatedeffects of the expression of the biphenyl pathway and asso-ciated genes in Burkholderia xenovorans LB400. Biodeg-radation 21: 147–156.

Pérez-Pantoja, D., Donoso, R., Junca, H., González, B., andPieper, D.H. (2009) Phylogenomics of aerobic bacterialdegradation of aromatics. In Handbook of Hydrocarbonand Lipid Microbiology. Timmis, K.N. (ed.). Berlin,Germany: Springer-Verlag Berlin Heidelberg, pp. 1355–1397.

Pieper, D.H., and Seeger, M. (2008) Bacterial metabolism ofpolychlorinated biphenyls. J Mol Microbiol Biotechnol 15:121–138.

Rahbarizadeh, F., Rasaee, M.J., Forouzandeh, M., Allameh,A., Sarrami, R., Nasiry, H., and Sadeghizadeh, M. (2005)The production and characterization of novel heavy-chainantibodies against the tandem repeat region of MUC1mucin. Immunol Invest 34: 431–452.

Saerens, D., Kinne, J., Bosmans, E., Wernery, U., Muylder-mans, S., and Conrath, K. (2004) Single domain antibod-ies derived from dromedary lymph node and peripheralblood lymphocytes sensing conformational variants ofprostate-specific antigen. J Biol Chem 279: 51965–51972.

Saerens, D., Ghassabeh, G.H., and Muyldermans, S.

(2008a) Single-domain antibodies as building blocks fornovel therapeutics. Curr Opin Pharmacol 8: 600–608.

Saerens, D., Stijlemans, B., Baral, T.N., Nguyen Thi, G.T.,Wernery, U., Magez, S., et al. (2008b) Parallel selection ofmultiple anti-infectome nanobodies without access to puri-fied antigens. J Immunol Methods 329: 138–150.

Sambrook, J., and Russell, D.W. (2001) Molecular Cloning: ALaboratory Manual, 3rd edn. Cold Spring Harbor, NY, USA:Cold Spring Harbor Laboratory Press.

Schneider, T., and Riedel, K. (2009) Environmental proteom-ics: analysis of structure and function of microbial commu-nities. Proteomics 10: 785–798.

Tecon, R., Beggah, S., Czechowska, K., Sentchilo, V., Chro-nopoulou, P.M., McGenity, T.J., and van der Meer, J.R.(2010) Development of a multistrain bacterial bioreporterplatform for the monitoring of hydrocarbon contaminantsin marine environments. Environ Sci Technol 44: 1049–1055.

Velázquez, F., de Lorenzo, V., and Valls, M. (2006) Them-xylene biodegradation capacity of Pseudomonas putidamt-2 is submitted to adaptation to abiotic stresses: evi-dence from expression profiling of xyl genes. EnvironMicrobiol 8: 591–602.

Wicker, J., Fenner, K., Ellis, L., Wackett, L., and Kramer, S.(2010) Predicting biodegradation products and pathways:a hybrid knowledge- and machine learning-basedapproach. Bioinformatics 26: 814–821.

Yang, H.-C., Cheng, J., Finan, T.M., Rosen, B.P., and Bhat-tacharjee, H. (2005) Novel pathway for arsenic detoxifica-tion in the legume symbiont Sinorhizobium meliloti.J Bacteriol 187: 6991–6997.

Supporting information

Additional Supporting Information may be found in the onlineversion of this article:Fig. S1. Alignments of bacterial thioredoxin-dependent ArsCspecimens. Ppu1928 and Ppu2716 correspond to ArsC1 andArsC2 proteins of P. putida KT2440 respectively. ArsC of B.subtilis(Bsu2578) and S. aureus(Sap0018) were used asthioredoxin-dependent model proteins in the alignment. Aperfect match in the catalytic residues (3-cys pairs in yellowand 1-arg in red) is observed in all cases.Fig. S2. Primary amino acid sequences of proteins used inthe immunization cocktail.Fig. S3. Primary amino acid sequences of VHH domainsthat bind strongly each of the proteins of the immunizationcocktail.Fig. S4. Phylogeny of the VHH domains with a strong bindingto each of the proteins of the immunization cocktail. Theanalysis was made on the primary sequences of VHH domainsshown in Fig. S3. Green and red squares highlight VHH

against mono-component and multi-component enzymesrespectively.Fig. S5. Evolutionary relationship between the proteins of theimmunization cocktail as deduced from the phylogeny of theirprimary amino acid sequences. The phylogram tree showspossible connections between the proteins used to inoculatethe camel. The graph was generated with the default settingsof clustalW algorithm.

14 O. Zafra et al.


Fig. S6. Comparison of primary sequences of inhibitory (F,R)and non-inhibitory (M,X) VHH domains targeting BphC ofB. xenovorans LB400. Conserved frameworks (FR) andcomplementarity determinant regions (CDR) are shown.Residue 30 of CDR1, which makes a difference in the struc-turing of the corresponding loop, is caged in red.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for thearticle.



Monitoring biodegradative enzymes with nanobodies raised in Camelus dromedarius with mixtures of...

Documents

Transcript of Monitoring biodegradative enzymes with nanobodies raised in Camelus dromedarius with mixtures of...