The Xanthopsins: a new family of eubacterial blue-light photoreceptors - 16944y

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The Xanthopsins: a new family of eubacterial blue-light photoreceptors

Kort, R.; Hoff, W.D.; van West, W.S.; Kroon, A.R.; Hoffer, S.M.; Vlieg, K.H.; Crielaard, W.; van Beeumen, J.J.; Hellingwerf, K.J. Publication date 1996 Published in EMBO Journal Link to publication

Citation for published version (APA):

Kort, R., Hoff, W. D., van West, W. S., Kroon, A. R., Hoffer, S. M., Vlieg, K. H., Crielaard, W., van Beeumen, J. J., & Hellingwerf, K. J. (1996). The Xanthopsins: a new family of eubacterial blue-light photoreceptors. EMBO Journal, 15(13), 3209-3218.

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TheEMBO Journal vol.15 no.13 pp.3209-3218, 1996

The xanthopsins:

a new

family

of eubacterial

blue-light photoreceptors

R.Kort,

W.D.Hoffl,

M.Van West,

A.R.Kroon,

S.M.Hoffer, K.H.Vlieg, W.Crielaard,

J.J.Van

Beeumen2

and

K.J.HellingwerfW

Department ofMicrobiology, E.C.Slater Institute, BioCentrum,

UniversityofAmsterdam, NieuweAchtergracht 127, 1018WS

Amsterdam,The Netherlands and2DepartmentofBiochemistry,

Physiology andMicrobiology, Laboratory of Protein Structure and

Function, StateUniversityofGhent, Ledeganckstraat 35, 9000 Ghent,

Belgium

'Presentaddress: DepartmentofMicrobiology andMolecular Genetics, Health Science CenteratHouston, University ofTexas,

6431Fannin, Houston, TX77030, USA 3Correspondingauthor

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic

photo-trophic purplebacteria.The PYP from

Ectothiorhodo-spirahalophila BN9626 is the only member for which the sequencehas beenreported at the DNAlevel. Here we describe the cloning and sequencing of the genes

encodingthe PYPsfromE.halophila SL-1 (type strain) andRhodospirillum salexigens. Thelatter protein

con-tains, liketheE.halophila PYP,thechromophore trans

p-coumaric acid,as we show here with high

perform-ance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacteriuminwhich this

photoreceptor has been identified. An ORF down-stream of thepyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis ofthechromophore of PYP. Thepyp gene

fromE.halophilawasusedfor heterologous

overexpres-sion inboth Escherichia coliandR.sphaeroides, aimed

atthedevelopment ofaholoPYPoverexpressionsystem

(an intact PYP,containing thep-coumaric acid

chromo-phore and displaying the 446 nm absorbance band).

In both organisms the protein could be detected immunologically,butits yellowcolor was notobserved.

Molecular genetic construction of a histidine-tagged version ofPYPled to its 2500-fold overproduction in

E.coliand simplified purification oftheheterologously produced apoprotein.HoloPYP could be reconstituted by the addition ofp-coumaric anhydridetothe histidine-tagged apoPYP (PYP lacking its chromophore). We

propose tocall thefamilyofphotoactive yellow proteins

thexanthopsins,inanalogywith therhodopsins. Keywords: Ectothiorhodospira halophila/photoactive yellow

protein/Rhodobacter

sphaeroideslRhodospirillum

salexigens/xanthopsins

Introduction

Thephotoactive yellow proteins (PYPs) constitute a new

family of eubacterial

photoreceptor proteins

(Hoff

et

al.,

1994b). Members have been isolated from the halophilic

phototrophic purple eubacteria Ectothiorhodospira halo-phila (Meyer, 1985), Rhodospirillum salexigens (Meyer et al., 1990) and Chromatium salexigens (Koh et al.,

1996). PYP is the first eubacterial photoreceptor to be characterized in detail and has recently been shown to contain a unique chromophoric group: thiol ester linked p-coumaric acid (Baca et al., 1994; Hoff et al., 1994a).

This is the first demonstration of a co-factor role for

p-coumaric acid in eubacteria, previously only known from higher plants (Goodwin and Mercer, 1983). The pathway of biosynthesis of p-coumaric acid has been extensively studied inhigher plants (Hahlbrock and Scheel, 1989), but no information is available on the conservation of this pathway in E.halophila or other eubacteria. In higher plants, the two enzymes of central importance in the metabolic conversions relevant for p-coumaric acid

are:phenylalanine ammonialyase (PAL),whichcatalyses

the reaction from either phenylalanine or tyrosine to

p-coumaricacid,andp-coumaryl:CoA ligase(pCL),which activates p-coumaric acid through a covalent coupling to CoA, via a thiol ester bond (Hahlbrock and Scheel,

1989).

The PYP from E.halophila is by far the best-studied memberof this photoreceptor family. Its crystal structure has recently been redetermined at 1.4 A resolution and shows that theproteinhasanoc/d fold, resembling (eukary-otic) proteins involved in signal transduction (Borgstahl etal., 1995). Evidence has been obtained indicating that PYP functions as the photoreceptor for a new type of

negative phototaxis response (Sprenger et al., 1993). Absorption of a blue photon

(Xmax

= 446 nm) induces PYP to enter a cyclic chain of reactions (Meyer et al., 1987). This photocycle involves two intermediates and

strongly resembles the photochemistry ofthe archaebac-terial sensory rhodopsins (Meyer et al., 1987; Hoff

etal., 1994c).

Recently, the ORF encoding PYP from E.halophila

BN9626 was cloned and sequenced (Baca et al., 1995).

Here wereport thecloning and thecomplete sequence of thepyp genes fromE.halophilaSL-1 (thetypestrain) and Rs.salexigens, which is the first gene cloned from this

organism, through reversegenetics. Directly downstream of the pyp gene inE.halophila welocated a geneencoding

aCoAligase homolog, suggestingaplant-likeconversion ofp-coumaric acid to its CoA derivative before linkage

toPYP lacking its chromophore (apoPYP).

Previously, we have reported the presence ofa single cross-reacting protein in a large number of eubacteria,

with a highly specific polyclonal antibody against PYP

(Hoff et al., 1994b). Here we report,

using

heterologous

PCRtechniques,theidentification ofa newPYP

homolog

in thegenetically well-characterized Rhodobacter

sphaer-oides. This finding opens the way to molecular

genetic

Sphl BarnHI EcoRI Sphl BainHi EcORI

1 kb -'

".1

PstI &9TaIPvull PvulI SMnai PvtAI PvtlC~dkc, PAl

I I I I dada pyp 500bp

pc/

* > -> 0 4- .4 - 4 -4- .

*4

4 - 4-4 4 CAGCTGGAGGAGCGCACCTGCGACCTCGATCCCGCCCCGTACTGGCTCCAA Q L E E R T C D L D P A P Y W L Q R Y * 4 4 GGCTGTACATCTAGGCTGGAGTCCCGAGAGTGAGCAAGGCTCACCACGAAGCCCCCGGTCCATGAAAGGAGTATCACGATGGAACACGTA GCCTTCGGTAGCGAGGACATCGAGAACACCCTCGCCAAGATGGACGACGGCCAGCTCGACGGCCTGGCCTTCGGCGCCATCCAGCTCGAC A F sS E D I E N T L A X M D D G Q L D G L A F G A I Q L D GGCGACGGCAACATCCTTCAGTACAACGCCGCGGAGGGCGACATCACCGGCCGCGACCCGAAGCAGGTCATCGGCAAGAACTTCTTCAAG G D G N I L Q Y N A A E G D I T G R D P K Q V I G X N F F X GACGTGGCCCCGTGCACTGACAGCCCGGAGTTCTACGGCAAGTTCAAGGAAGGGGTGGCCTCGGGCAACCTGAACACGATGTTCGAGTAC D V A P C T D S P I F Y GK F R E G V A S G N L N T K F E Y ACCTTCGATTACCAAATGACGCCCACGAAGGTGAAGGTGCACATGAAGAAGGCCCTCTCCGGCGACAGCTACTGGGTCTTCGTCAAGCGC T F D Y Q K T P T K V K V E K K K A L S G D S Y W V F V K R GTCTAGACCAGACCCCGCTGCCCTGTCCCaOCCGa=CGCGAAAGGACCGACCGATGCAAGGGCTGAATGCCGATGAAGT V * Q G L N A D Z V GCTGCGGTTGCTGCGCAGCCTGATCCCCGGCGAGCTGGCCACCGGCCGCGGTCAACGGGGCGACCCGCCCGAAGCGCAGGACCTGTGCGC L R L L R S L I P G E L A T G R G Q R G D P P E A Q D L C A CGATAGCCGCCTGGATGCCGAACCAATCCGGGCGGACTCCCTGGATCGACTCAATCTAGCCAGCGCGCTCAACCoCTTCTTCCGCCTGCA D S R L D A E P I R A D S L D R L N L A 8 A L N R F F R L S 4 TGAGACCGGTGTGGAGGACCGTCTGCTGGCCGTGCGCCGGATCGGCGACATGGCCGAACTCATCGCCGACGCCAGTCAGCACACCAGCQG E T G V E D R L L A V R R I G D N A F L I A D A 8 Q E T 8 G CCTGACCTCTCGACCTCaGGCAGCACaGGCACCCCGCAGCCGCACCACCACAG CGGCGCACCCAGGAGGCCGAGGCACTGGC L T F S T S STGT P Q P E S W A A L T Q A E A L A CAACGCCCTGCAGTCACCCGCGGGTGATCGCCTGGCTGCCCGTCCACCACCTCTACGGTTTCGTCTTCGGCGTCGCCCTGCCCCGCGC N A L O S I P R V I A W L P V E E L Y G F V F G V A L P R A GCTGGGCAGCACCGTGATCGAGAGCCACGCCGCGCCCACCGCCCTGTTCCGCGAGCCGGCACCTGACGACCTOATTGCCACCGTCCCGGC L G S T V I Z SB A A P T A L F R E P A P D D L I A T V P A ACGCTGGCGTTACCTGTTCGATAGCAATCACCGCTTCCCCGGCGGCACGGGCATCAGCTCGACCGCCGCGCTGGAGACCGCCTGCCGCAA R W R Y L F D S NK R P P 0 G T 0 I S S T A A L E T A C R N CGGGCT TTOCAGGCCGGCCTGGACGCTCTGCTGGAGGTCTACGGCCCCACCGAGGCCGOCGATCGGCCTGCGCG CCCTCGGA

G L L Q A G L D A L L E V Y O A T Z A o G I o L R W A P S E GGACTACCGCCTGCTOCCCCACTGGCACGGCGACGCOACGGCAACCTCCAGCGCACTCAATCCCGAT(MTGCAGCGGTGACCOTGGCCCC D Y R L L P E W GO D A T A T 8 S A L N P D G A A V T V A P GCTCGATCGCCTCCAGTAGCGGGACGAGCGG GTCTTCCG ACCCACC L D R L Q W R D ER V F R P T O R I D D I Q I GO V N V 8 TCCaGGaCACGTCGCGCGGCGCCTCGAAAGCCACGAG CCGTCCTCGCGGTCCGCAGCCACGAAGGCAGTCOTCGGCGCCT P GO V A R R L S8 I I A V A A C A V R 8 OG a 8 R R R L

GAsAGCGTTCA TT TCCGACCGATC _CTACCGGC

R A F I V P A R 8 D A D P E T L R Q T L E N W I W EZ L P A

GGTCGAGCGACCCACGOATCTGCGTATCGG CACCOAGCTTCC COCAGCAACGCGAG

V E R P T D L R I G T E L P R N A K G K L Q 90 180 270 360 450 540 630 720 810 900 990 1080 1170 1260 1350 1440 1530 1620 1710 1777

Fig. 1. ThepypgenefromE.halophilaSL-I withflankingregions. (A) Physicalmap of the chromosomalregioncontainingthepypgene.The

cloned2.4 kb PstIfragment,which is locatedonthe 5.2 kbEcoRI-SphI fragment, isshown indetail,indicatingthepositionof thedada, pypand

pclgenes.Theopenarrowindicates the direction of thegenes.(B)DNAsequenceof the 1.8 kbPvuII-PstIfragment containingapartial ORFJ,the

E.halophilapypgeneandapartial ORF3. Thederived amino acidsequencesaregiven atthe firstpositionof eachcodonbytheone letter code. The

stopcodon isindicatedbyanasterisk. TheputativeAT-rich promoterregion (41 mol%GC)isunderlined. Putative ribosome bindingsitesaredoubly

underlined andan inverted repeat is overlined. Underlined amino acidsarepart ofahighlyconservedmotif inAMP-bindingproteins(Fuldaetal., 1994). The bases indicated byavertical arrowdifferfrom theformerly published Ehalophila BN9626sequence(Bacaetal., 1994).

studies ofthe function ofPYP. TheEhalophilapypgene was heterologously overexpressedinEscherichiacoliand R.sphaeroides, yielding (mainly) apoPYP.Thepurification

of a histidine affinity-tagged derivative of PYP from E.halophila, overproduced in E.coli, yielded a 2500-fold

overproduction of apoPYP. Intact PYP, containing the p-coumaric acidchromophoreanddisplaying the446nm

absorbanceband(HoloPYP)could bereconstitutedbythe addition of p-coumaric anhydride to the recombinant

apoPYPasdescribed forapoPYP (Imamotoetal., 1995).

These results will facilitate detailed biophysical studies

on a protein with a unique set of characteristics: it is

water soluble, photoactive and its structure is known at

1.4 A resolution. R.Kort et al

A

B

1 91 181 271 361 451 541 631 721 811 901 991 1081 1171 1261 1351 1441 1531 1621 1711

Xanthopsins: genesandoverexpression SaA 1 kb I PvA

llp

Rlali

Pstl Ecdo 1 500 bp I pyp

*~~~

> . -< --4 - 4 4 -CGATCGCCTCCTCGGCTCCAGAACCAGGCGATGGCGCAGATAGGGGCTCAGGTGaCAGACGCTGTCGTCGGCATCGGGACCGTAATCGGT GTTGCGGTcGGCCGCATAGATCCOCCCGGCCCGGGGCGCGAAGGcGTCGAGCcGGGCCAGGCCAGcGGcGCGGGTCGTCCGCTGGCoTCT OGGGTGAATCGGCGTGCGCTGGCGCGACCGCTTCGACGCGGGAGGCTTCAATCATGATGGCGGCGGTTCCTTGGCGGCGGTGCGAGCCTG CCGGGCGCTGCGCCGGCAGCGTTCCGAGCAATAGCGCACGCGCTCCCAATCCCGGGCCCACTTCGCCAGGTGA&0GGGCGGTT0CA TTCGGCGCCTTGTTTTTGG&GGTCGGCCTTTTTGCGC&TGAcGGGGTTTcGATGTGACCACATGGTATCCGGTTCGTGCCAACCGGG GCCCCGGATCTGCCCCAGTTGCACTCGCTGGCGCGATGCGACAATGTGGCCGACCGGCAGGCGAGGGAGGCCGACAGCGCTGCAGCACAG GGCGGCGGCGGACGTGACGGCCCACCGCAGCGACACGACGAACCGTTGACCTCTTGCGTCCGGTCCCCACaAAGCCCGGC

CACCGGGQTTTTTATC ATTGTA~TAAA~GGGCGCAC CCGTCAC

aGaAaACTTGCTAT<}aAAATaATCAGGACGACATCGAGAACGCCATG

ATATaGGCGACGCGCAGATCGACGACCTG --MEX" I K F aQ D D I F N A X A D M G D A Q I D D L GCTTTTGCGCCATTCAACTGGACGAGACCGGCACGATCCTGGCCTATAACGCGGCCGAWGCGAACTlACCGGCCGCAGTCCCCAAGAC A F G A I Q L D N T G T I L A Y N A A F G E L T G R S P Q D GTGATCGGCAAGAACTTCTTCAAGGACATAGCGCCGTGCACCGACACCGAaGiAATTCGOCGGCCGGTTCCGCGAAGGTGGCCAATGGC V I G K N F F K D I A P C T D T E E F G G R F R E GV A N G GACCTGAACGCGATGTTCGAATATGTCTTCGACTATCAGATGCAGCCGACCAAGGTGAAGGTGCACATGAACGCCCTATCACCGGCGAC D L N A N F Z Y V F D Y Q X Q P T K V K V H X K R A I T a D

AGCTACTGGATCTTCGTCAAGCGTGTCTGATCCTCTCGACGCCCCAGCACCCT _TCGCCHTCa T0Ca CCGCGA

S Y W I F V K R V *

TlsGCTGsTCCGGiTaaTCGCTGATGTIOTGGCG CCCCGCGATGC TGCCGCCTT4TCCGACGACACGCCGCTGAC

CGATGCGGCGGGGTCGGGTGACGGACCTGGGCTCGACTCGCT CCTCGTCAATGCOOCCAGCGACCTGACCCGGCGCCTGCCTGGA

CGAGAGTGrGCCAGGAGGAACGCTTGCTGCGCCAGCGCACGTTCCCACACTGGGTCGAC

Fig. 2. Thepvpgenefrom Rs.salexigens with flanking regions. (A) Detailed physicalmapofthe cloned 1.4 kb PvuI-SalIfragment from

Rs.salexigens,indicating the position of thepyp gene.Theopen arrow indicates the direction of thegene. (B) DNAsequenceofthe 1.4 kb PvuI-Sall chromosomal fragment fromRs.salexigens containing thepvp genewithflanking regions.The derived amino acidsequenceisgivenatthe first

position of each codonby theone letter code. Theputative AT-richpromoterregion (35mol% GC)isunderlined. Theputativeribosome bindingsite

isdoubly underlined.

Results

Thepyp genesfrom EhalophilaandRs.salexigens The DNA sequence ofa 1.8 kb PvuII-PstI fragment was

determined (Figure IA) and is shown in Figure lB. The

amino acidsequenceofE.halophila PYPpredictedonthe basis ofthis sequence information is identical to the one

determined by amino acid sequencing (Van Beeumen etal., 1993),exceptforposition 56 which isaGln instead

of a Glu, as also observed in the DNA sequence of the

pyp gene from E.halophila BN9626 (Baca etal., 1995).

A potential AT-rich (41 mol% GC) promoter region can

beidentifiedupstreamoftheORFencodingPYP(positions

60-103, Figure IB), which may be essential for the

formation ofan open complex for initiation of transcrip-tion. Also, a potential ribosome binding site (RBS) is

located directly upstream of the PYP ORF. Directly downstreamof thePYPORFaninverted repeat is located

(positions 557-587, Figure iB).

ThepypgenefromRs.salexigensis the firstgenecloned

from this bacterium. It was localized on a 1.4 kb

PvuI-Sall chromosomal fragment. Sequence analysis of this

fragment (Figure 2B) showed that it contains the entire

ORF encoding PYP; the predicted amino acid sequence

contains 125 amino acids and completely matches the amino acid sequence of this protein (Koh et al., 1996). Upstream of theORF, apotential AT-rich (35 mol% GC)

promoter region (positions 638-680, Figure 2B) and ribosome binding site can be recognized, while directly downstream of the ORF an inverted repeat is present

(positions 1134-1164, Figure 2B).

Identification ofa PYPhomologin R.sphaeroides

Chromosomal DNA fromR.sphaeroides 2.4.1. was used

as template in a PCR with two primers homologous to conservedpyp sequences to yield a 0.3 kb product. The

validity ofthe PCR product was confirmed by Southern

hybridization experiments with R.sphaeroides chromo-somal DNA under stringent conditions, using the PCR fragment as a probe. This revealed strong and specific hybridizationsignals (datanotshown).The DNAsequence

of the productshowed that theencoding proteinsequence

was homologous to PYPfromE.halophila, Rs.salexigens

and Chromatium salexigens (Figure 3). Comparisonof PYPsequences

The complete amino acid sequences of the PYPs from

E.halophila, Rs.salexigens and C. salexigens (Kohetal.,

3211

A

B

1 91 181 271 361 451 541 631 721 811 901 991 1081 1171 1261 1351 90 180 270 360 450 540 630 720 810 900 990 1080 1170 1260 1350 1408

F

m I

R.Kort etal. 0 0 Chromophore Tyr42 s

070H

-/ Protein N' t.go 0 )CNsO~4 HO N ON

N

O N N rTr 9 70 N L

LQYNgAEGDITGRD:)P!KQVIGKNFFKDVAPCTDSPEFYXKSG KEGVASGNL,NTMPMFEYTIFDYQ..

...A; $EELTGRSPQD KDIGMCKt CDTEENFGRO

REGVANDLNAAE:YvFDYQ

.

.Q>G I TGt2KSVF FKD SC K Q GRH ES SNLATM YV-FDYQ.

*~ ~ ~~TG K NG

e.LKY, GI,PANRADVTGNi;F-VNE IICAKGKRiHGELT.LFHQPGQVNVMFDYKFAYK.

# # # 4

Fig. 3. Sequence conservationinthefamily of photoactive yellow proteins: the xanthopsins. Model for the p-coumaric acid binding pocket basedon

crystallographic data (Borgstahletal., 1995) andsequenceconservation of the residuesforming this pocketin the PYP sequencesfromEhalophila, Rs.salexigens,C.salexigens, and R.sphaeroides. Sequence conservation is indicatedin gray,with themoreand lessessential residues forp-coumaric acidbinding indicatedinblue(asterisks)and orangerespectively. The unique Cys69, which bindsthechromophore,is indicated ingreen, the

chromophoretransp-coumaric acidand the thiol esterlinkage in yellow.

1996) are homologous, with 66% of the amino acids identical in all three sequences. This result enabled us to

obtain the partial sequence of a PYP homolog from R.sphaeroides (see above). A partial alignment of these four sequences is shown inFigure 3. Allproteins contain the Cys residue that in the E.halophila protein has been shown to bind covalently to the chromophore (Van Beeumen et al., 1993). From the 1.4 A crystal structure

ofPYPitcanbe concluded thatTyr42, Glu46,Arg52and

to a lesser degree Thr50 and Tyr98, in the E.halophila

PYP, are important for the protein-chromophore inter-actions that lead to the deprotonation of the p-coumaric

acid molecule and result in the tuning of the absorbance ofthis cofactor to 446 nm (Baca et al., 1995; Borgstahl

et al., 1995; Kim et al., 1995). These residues are all conserved in the PYPs from E.halophila, Rs.salexigens

and

C.salexigens

(Figure 3), in line with the similarities between these proteins with respect to their absorbance spectrum and photochemical properties (Meyer, 1985; Meyeretal., 1990). In thesequence of theR.sphaeroides

PYP homolog these six residues, of central importance

for the binding of the chromophore, are also conserved, with the exception ofThr 50 (Figure 3). Furthermore, a strongconservation is observed in the sequence VIGKNFF, which forms a type IItightturnbetween thea4-helix and the

P3-strand

ofPYP (Borgstahl et al., 1995).

Analysis ofpyp flanking regions

The 1.8 and 1.4kb chromosomal fragments from E.halo-phila and Rs.salexigens respectively, were examined for the presence of ORFs. In addition to the PYP ORFs

presented above, this analysis indicates the presence of a

large partial ORF (391 residues) downstream of the pyp

gene from E.halophila (Figure 1B). This ORF was not

found in the chromosomal fragment from Rs.salexigens.

In line with this, comparison of the 1.8 and 1.4 kb chromosomal fragments from E.halophila and Rs.salexi-gens showed that the sequencesimilarityin thesefragments

is confined to the ORFs encodingPYP.

Upstream of the pyp gene from E.halophila SL-1 an

ORF is located that shows significant homology to the E.coli dada gene, encoding the small subunit of the membrane bound iron-sulfur flavoenzyme D-amino acid

dehydrogenase (Olsiewski etal., 1980), as was found in

E.halophila BN9626 (Baca etal., 1994). The partial ORF downstream of thepyp gene fromE.halophilawasfurther analyzed by searching for sequence similarities with

proteins in the SwissProt database. The most similar

proteins were foundtobe anumberof CoAligasesfrom variousorganisms with ~24% sequenceidentity and48%

similarity over a stretch of 400 amino acids (Table II).

Furthermore,thisputative pclgene(seeFigure IA) shows,

like thepyp gene, a high GC-bias in the wobble position of its codons, which is indicative of its functionality. In

Rs.salexigens the ORF encoding a CoA ligase homolog has not been found downstream from the pyp gene. This may suggest a larger intergenic region between pyp and the putativepcl in this latter organism. This is supported

byaSouthern blot, showing hybridizationofRs.salexigens

chromosomal digests with the putative E.halophila pcl

(M.K.Phillips-Jones, unpublished observations).

Identification of the chromophoreofRs.salexigens

pyp

The chromophore of Rs.salexigens PYP was identified

high-Xanthopsins:genes andoverexpression

A

B

A 1~~~~~~~~~~~~~~~~~~~1 a U elutiontine(mh) A 0~~~~~~~~~~c I0 I0 m)~ ~~~ ~~~~~~~~~~~~~~l 11%, :3 .9 0 9 I a

elutbon time(mi)

D

,d

eluHontlme(rin)

Fig. 4. Identificationof the Rs.salexigens chromophore withcapillary electrophoresis. (A) Electropherogram of ethyl acetate extract from

solubleprotein fraction of anaerobicallygrown Rs.salexigens;

p-coumaricacidelutesat 10min. (B) Electropherogram ofp-coumaric

acid,predominantly thetransisomer(Sigma).(C)Electropherogram ofextractedchromophore fromanaerobically grownRs.salexigens

co-injected withp-coumaric acid, showing an increase of thep-coumaric

acidpeakat 10min. (D) Electropherogram ofextractedchromophore from aerobically grownRs.salexigens.

performance capillary zone electrophoresis (data not shown), which uses the electrophoretic mobility of ions

as separation principle (for a review see Karger et al., 1989). After injection and electrophoresis of an ethyl

acetate extract from the soluble protein fraction of

Rs.salexigens, theelectropherogram shows a major com-ponent at 10 min (Figure 4A), in an amount of 0.1 pmol

ofp-coumaric acid (see Materials and methods), which

corresponds with8 pmol of detectedp-coumaric acid per mgsolubleprotein.Asacontrol,co-elution of p-coumaric acid (Figure 4B) with the chromophore in the extraction mixture was demonstrated by the increase in size of the

peak at 10 min (Figure 4C). Furthermore, our analysis shows thatnop-coumaricacid isbound to solubleproteins

in aerobically grown Rs.salexigens cells (Figure 4D), whichis independent proofof regulation of PYP expres-sion in thisorganism (compare Hoff et al., 1994b).

Heterologous overproduction oftheEhalophila Pyp

To overexpress PYP from E.halophila, a 0.45 kb AvaIl fragmentfrompYAMA958 containing thepyp ORF, was

inserted into the overexpression plasmid pT713 (Studier et al., 1990) to yield pTY13. After transformation of pTY13 to E.coliBL21, 50- to 100-foldoverproduction of PYPwasoberved using Western blots and rocket immuno-electrophoresis (RIEP). However, absorbance spectra of the cytoplasmic fraction of these cells do not show an

absorbanceband at 446 nm, while this band wasexpected to be clearly visible onthe basis of the concentration of PYPdetermined by RIEP(data not shown). This indicates that E.coli BL21/pTY13 mainly produces apoPYP, i.e. PYPwithout the chromophore.

In an attempt to obtain an overexpression system for holoPYP, the plasmid pART3 (see Table I), containing the same 0.45 kb insertwith thepyp gene from E.halophila, was conjugated to R.sphaeroides DD13. Since this organism is phototrophic, like E.halophila, and therefore produces a large arrayof pigments, it may also synthesize p-coumaric acid. The DD13 strain is mutated with respect to synthesis of the photosynthetic apoproteins (Jones

et al., 1992), reducing the absorbance of the associating pigments, thereby facilitating the observation of the expected absorbance band at 446 nm, causedby holoPYP. RIEP experiments showed that the transconjugant R.sphaeroides DD13/pART3 also produces PYP at levels

100-fold higher than E.halophila (data not shown). Approximately 50% of the PYP produced was associated with the membrane fraction from these cells. However, also in this case the expected absorbance band at 446 nm for holoPYP was lacking (data not shown).

A chimeric version of the pyp gene from E.halophila was cloned inE.coli,which allows one to isolate PYP by the presence of ahistidine affinity tag in the gene product and to confirm the lack of the chromophore in PYP produced in E.coli. Surprisingly, E.coli M15/pHisp (see Table I) overproduces PYP at levels of 50 mg/l culture per

OD660

unit,asdeterminedby RIEP (FigureSA),which is -2500-foldhigherthanE.halophilaand ~50-fold higher than in the case of the two overexpression systems described above.Cell-freeextractsfrom E.coliM15/pHisp were used in Ni-affinity chromatography. This method

yielded -75% pure protein in a single step (Figure SB). Incubation of the isolated histidine-tagged PYP with enterokinase yielded a product with a molecular weight

indistinguishablefrom nativeE.halophilaapoPYP (Figure

SB). The absorbance spectrum of the isolated

histidine-taggedPYPshows that the typicalabsorbance band in the visible region of the spectrum is completely lacking

(Figure SC). This indicates that the protein produced in this E.coli strain ishistidine-tagged apoPYP (HAP).

To demonstrate the usefulness of HAP for further

biophysical studies on PYP, we reconstituted HAP with

p-coumaric anhydrideinto holoPYP. Thefollowing observ-ations showed that reconstitution of holoprotein was

achieved: (i) spectral analysis showedanabsorption band

at446 nm, which increased (tosaturation)withastepwise

addition of the p-coumaric anhydride; (ii) analysis of absorbance spectra in time showedanincrease at446nm

and a decrease at 350 nm, in line with an increase of holoPYP concentration and a decrease of the anhydride concentration; (iii)purifiedreconstituted holoPYP showed

an absorbance spectrum like that ofpurified native PYP

(Figure SC); (iv) reconstitutedholoPYPcanbereversibly

bleached after absorption oflight (data not shown). The 3213 u 'ID I la N OD -N tl: 11 12

R.Kort etal.

TableI. Strains and plasmids used in this study

Strains and plasmids Description Source or reference

Strain

E.coli BL21 hsdS, gal,(Xclts857indl,Sam7, ninS, lac UV5-T7 gen 1) Studier and Moffat (1986) E.coliM15[pREP4] expressionhost with repressor plasmid, KmR Qiagen

E.coliTG1 supE,A(lac-proAB), hsdA5, F'[traD36, proAB+,laclJ,lacZAM 15] Gibson (1984)

E.coliS 17-1 RP4-2(Tc::Mu)(Km::Tn7), thi,pro, hsdR, hsdM+,recA, TpR, SmR Simon et al. (1983) E.halophila SLI typestrain Raymond andSistrom(1969)

R.sphaeroides 2.4.1 typestrain VanNiel (1944)

R.sphaeroides DD13 RC-, LHI-, LH2-, KmR, SmR Jones etal. (1992)

Rs.salexigens WS 68 typestrain Drews (1981

Plasmid

pCHB500 pRK415 andpSH3 derivative, TcR Benning and Sommerville

(1992)

pART3 0.45 kbE.halophilaAvaIl fragment cloned intopCHB500 this study

pQE30 RBSII,6XHis tag, ColEl ori, ampR Qiagen

pHisp 0.42 kbE.halophilaPCRproduct cloned intopQE30 this study

pT713 expressionvector,T7 promoter,AmpR GibcoBRL

pTY13 0.45kbE.halophilaAvaIl fragment cloned intopT713 thisstudy

M13mpl8/19 M13mpl derivedphages,lacZ' Messing and Vieira(1982) pYAMA18 2.4 kbEhalophilaPstIfragment clonedintoM13mpl8 this study

pYAMA958 1.8kbEhalophilaPvuII fragmentclonedinto M13mpl8 thisstudy pS16 1.4kbRs.salexigens Pvul-SalIfragmentcloned into M13mpl9 thisstudy

masses of the histidine-tagged holo- and apoPYP were

determined by ESMS to be respectively, 16.0081 and 15.8625 kDa. These values correspond well to the calcu-lated molecular weights of 16.0081 and 15.8611.

Discussion

Wereport here theDNAsequence oftwogenesencoding proteins known to be yellow and photoactive. The sequence of pyp from E.halophila SLI (type strain) is identical to the sequence reported for the pyp gene from

E.halophila BN9626 (Baca etal., 1994). In the flanking regions six differences between the two sequences were

found, which in five cases did not lead to changes in amino acid residues (see Figure iB); this indicates the close similarity but distinctness of these two strains.

Interestingly, all silent mutations are from T in the

E.halophila BN9626 strainto G or C in the E.halophila

SL-1 strain. This may be explained bya slightdifference inthe overall GC-content between the two strains, which have been isolated from different environments; the BN9626 strain was isolated from the Wadri Natrun, Lake AbuGabara nearBirHooker, Egypt(Imhoffetal., 1978) and the type strain SL-1 from Summer Lake, OR, USA

(Raymond and Sistrom, 1969). The GC-content of the cloned DNA fragments from E.halophila SLI and Rs.salexigenswascalculatedtobe67.3 and 65.8% respect-ively, which matches well with the overall GC-content from these organisms, being 68.4% (Raymond and

Sistrom, 1969) and 64 + 2%(Drews, 1981) respectively. The lack ofa signal peptide sequence upstream fromthe twopyp genes is in line with the intracellular localization ofPYP inE.halophila, as determined with immuno-gold

labeling experiments (Hoff et al., 1994b). Furthermore, the isoelectric points of the PYPs from E.halophila and

Rs.salexigens are predicted to be 4.63 and 4.23 respect-ively. ForE.halophilaPYP, this parameterwas

experiment-ally determined tobe 4.3 (McRee etal., 1986).

The sequence data for these two PYPs were used to

design primers for the amplification of a fragment from chromosomal DNA by heterologous PCR, leading to the identification of a PYP homolog in R.sphaeroides. The PCR product obtained was used as a probe to clone the R.sphaeroides pyp gene. This gene encodes a protein

of 124 residues, which cross-reacts with a polyclonal

antiserum raised against E.halophila PYP (data not

shown). The amino acid sequence of the R.sphaeroides

PYP homolog is ~46% identical to the sequence of the PYPs from E.halophila, Rs.salexigens and C. salexigens,

indicating that this PYP belongs to a different sub-group of the yellow proteins (R.Kort and S.M.Hoffer, unpublished observations).Since R.sphaeroides is

genetic-ally accessible, this opens up possibilities for genetic

studiesconcerningthefunction of PYP. The identification of this PYP homolog raises the question whether the

R.sphaeroidesproteinalso bindsap-coumaricacid chromo-phore. The conservation of Cys69, Tyr42, Glu46, Arg52

and Tyr98 in the R.sphaeroides sequence suggests that this may indeed be so. This leads to the prediction that

R.sphaeroides, in addition to its well-studied positive phototactic and chemotactic responses (for a review see Armitage, 1992), displays additional phototaxis response(s), based on PYP (see Sprenger et al., 1993). Thisprediction is currently being tested.

Directly downstream of the pyp gene from

E.halophila

an ORF is located that shows the highest sequence similarity to a range ofCoA ligases (Table II), including

p-coumaryl-CoA ligases. The putative E.halophila CoA

ligasecontains the motif TSGSTGTP (Figure iB), which is conserved in all members of the AMP-binding protein

family,of which thecoumaryl-CoAligases formadistinct subfamily (Fulda et al., 1994). This motif resembles the knownloop-formingadenine-binding motif (Saraste et al., 1990).Inplants, coumaryl-CoA ligase is of central

import-ance in the metabolism of p-coumaric acid (Hahlbrock and Scheel, 1989). This suggests that in Ehalophila,

p-coumaric acid is likewise activated by the formation of

Xanthopsins:genesandoverexpression

A

j ,4.

XoOOO.

t

1

2

0

20

40

60

10

30

50

120

Tind

1

2

3

4

5

205-116 80_ 49 32- 27-18 - 6-0.5 --- reconstitutedHAI' _---- HA%P 0.4 r 0.2-0.1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0.1-

~~~//

" / O-- -250 300 350 400 Wavelength(nm) 450 5

esterification was demonstrated by the fact that in vitro reconstitution of holoPYP was observed with the thio-phenyl esterofp-coumaric acid and notwithp-coumaric

acid (Imamoto et al., 1995). A further indication for a

functional coupling of the pyp andpcl gene products is the presence of an inverted repeat between these two

codingregionsand the absenceofarecognizablepromotor sequence, directly upstream of the pcl gene (see

Figure

iB). This indicates that transcription of the pcl gene occurs by readthrough of this inverted repeat from the promoter directly upstream of the pyp gene.

The biosynthesis ofp-coumaric acid, which in plants

can be performed inone step by phenylalanine ammonia lyase (Hahlbrock and Scheel, 1989),may consist of three consecutive steps in prokaryotes (compare the amino acid fermentation scheme of the anaerobic bacterium

Clostridium sporogenes; Bader et al., 1982). If so, an

aromatic aminotransferase, a 2-keto-acid reductase and a

dehydratase respectively, would be involved. In the first reaction, pyruvate may be the amino acceptor, as shown for manyaminotransferases. Thereformation ofpyruvate would then be carried out by alanine dehydrogenase. Interestingly, the dada gene upstream of the pyp gene (Figure IA), encodes an alanine dehydrogenase.

Based on the observations described above, one can

conclude that the organizationof the genes encoding the PYP sensory system is completely different from that of the only other well-studied class ofbacterial

photorecep-tors: the archaebacterial sensory rhodopsins. For sensory

rhodopsin I (SR-I) it has recently been shown that

tran-scription of the

sopI

gene (encoding theSR-I apoprotein)

is transcriptionally coupled to an ORF immediately

upstream ofthe sopI gene; this upstream ORF (the htrI

gene) encodes the signal transducerinteracting with SR-I (Yao and Spudich, 1992;

Ferrando-May

et al., 1993; Spudich, 1994).

In the soluble protein fraction of Rs.salexigens cells,

we could detect the PYP chromophore

p-coumaric

acid (8

pmollmg

solubleprotein). Thisfindingmadeaprotocol

available forstraightforward screening of intact cells for the presence of this chromophore. This may be of great importance, since the nature of thechromophorein

recep-tors for a largenumber ofblue-light responses, observed in microorganisms as well as in plants, has not yet been elucidated (Senger, 1987). The amount of chromophore identified in Rs.salexigens is equivalent to 0.1

jIg

PYP

per mg solublecellprotein, similartothecellularcontent

of PYP in

E.halophila

(Meyer etal., 1985).

We propose todesignate thefamily of PYPs 'xanthop-sins', which is derived from the Greek words

=avOo;

Fig.5.Overproduction, purificationand in vitroreconstitution of histidine-tagged PYP. (A)RIEPanalysis ofPYPproductionin Ecoli

M15/pHisp after induction withIPTG.Wells 1 and 2 contain solutions ofpurifiedPYPfromEhalophilawith knownconcentrations;the

followingwellscontain cell materialfromEcoliM15/pHisptakenat

the indicated induction times(T,nd inmin) after the additionof IPTG.

(B) SDS-PAGE of cell-freeextracts from EcoliM15/pHisp (lane 5),

histidine-taggedPYPisolated from thisextractbyNiaffinity

chromatography (lane 4),thesamepreparation after5h(lane2)and 24 h(lane3)of incubation withenterokinase,and PYPpurifiedfrom

Ehalophila(lane 1).(C)Absorbance spectrum of thehistidine-tagged 00n PYP (HAP) isolated from EcoliM15/pHisp and thespectrum of HAP

after reconstitutionwiththep-coumaricanhydrideandsubsequent purification. 3215

B

MVW(KDca) 0.3 0 n 0 / i! II i; i!

R.Kort etal.

Table II.Homologyofthe putative coumaryl-CoA ligase from Ehalophila with CoA ligases from other organisms

Enzyme (number ofamino acids) Organism Identity(%) Similarity (%) Reference

CoA ligase homolog(391) Ehalophila 100 100 this paper

Acetate-CoA ligase (660) A.eutrophus 25.1 49.5 Priefert andSteinbuechel (1992)

Acetate-CoAligase (672) M.soehngenii 20.6 47.5 Eggen etal.(1991) Long-chain-fatty-acid-CoA ligase (558) Ecoli 26.3 51.5 Black etal.(1992) Long-chain-fatty-acid-CoA ligase (700) yeast 22.8 47.5 Duronio etal.(1992)

Coumaryl-CoAligase (545) potato 22.4 45.5 Becker-Andre etal. (1991)

Coumaryl-CoA ligase (563) rice 25.3 49.2 Zhao et al. (1990)

Identity and similarity values are based onfulllength alignments made with the Genetics Computer Grouppackage program BESTFIT using a gap weightof3.0and a length weightof0.1.

(yellow) and

oift;

(eyesight). The bacterial xanthopsins resemble the archaebacterial sensory rhodopsins at the level of photochemistry (Hoff et al., 1994c), as well as offunction,which isproposedtobe that of aphotosensor in negative phototaxis (Sprenger et al., 1993). Further evidence for the xanthopsins, as a eubacterial protein family, has been obtained by studies withahighly specific polyclonal antiserum against E.halophila PYP, which showed the presence of a single, cross-reacting protein,

with a size of 15 kDa, in a large numberof prokaryotic microorganisms (Hoffetal., 1994b).

The results reported here define the xanthopsins as a

protein family of photosensors with strong sequence conservation andahighly conservedchromophorebinding

site. Inaddition,wehaveidentifiedagenethatmostlikely

encodesan enzymeinvolved inp-coumaricacid activation and that therefore is essential for in vivo holoPYP syn-thesis. Theheterologouslyproduced apoPYP wasusedas

substrate for in vitro holoPYP reconstitution, which is essential for further biophysical studies on intact and

directionally mutagenized PYP and for hybrid forms of PYP, containing chromophore analogs (A.R.Kroon and H.P.M.Fennema, unpublished observations). In addition, thediscovery ofaPYPhomologinR.sphaeroidesrenders this newphotoreceptor family genetically accessible.

Materials and

methods

Bacterial strains andplasmids

The strains and plasmids used in this study are listed in Table I.

E.halophila SL- 1,thetypestrain,wasobtained fromDeutscheSammlung

von Mikroorganismen und Zellkulturen (DSM), Braunschweig, strain number 244.

Cellculturing

E.halophilaSL- I(Raymond andCistrom, 1969) and Rs.salexigens WS68 (Drews, 1981)wereculturedphototrophicallyasdescribed (Meyer, 1985

and 1990respectively), unlessspecified otherwise.R.sphaeroidesstrain 2.4.1 (vanNiel, 1944)wasgrownaerobicallyin Luria Bertani broth.

DNAmanipulation

Chromosomal DNA was isolated according to standard procedures (Sambrooketal., 1989)fromEhalophila,Rs.salexigensand

R.sphaero-ides. All additional molecular genetic techniques were performed as

describedinSambrooketal. (1989).

Southern hybridization

Southern blots of chromosomal DNA from both Ehalophila and

Rs.salexigenswereprobed usinga94bp PCR productconsistingof an

internalfragment from the Ehalophila pypgene (seebelow).Theprobe

waslabeledwiththe Klenowenzymeby randompriming usingtheDIG DNA labeling kit and detected with Nitroblue tetrazolium salt, as

describedbythemanufacturer(Boehringer, Mannheim). Southernblots

of chromosomalDNAfromE.halophilaandR.sphaeroideswere

hybrid-ized at 65°C and washed at 65°C with 0.1x SSC buffer containing

0.1I% SDS. The blotscontaining chromosomalDNAfrom Rs.salexigens

were hybridized at 50°C and washed at500C with 0.5>x SSC buffer containing0.1% SDS.

Cloningof the E.halophila pyp gene

Pstl-digestedEhalophila chromosomalDNA wasused astemplatein a

PCR-reactionwith degeneratedoligonucleotides YS-Iand YS-2 with the sequencesAARAAYTTYTTYAARGAandGTCATYTGMTARTCRAA

respectively, as based on the PYPamino acidsequence (Van Beeumen etal., 1993). PCR wasperformed withthe enzyme Taqpolymerase(HT

Biotechnology,Cambridge, UK)for 30 cycleswith I min denaturation at94°C, I minannealingat20°Cand I minelongationat70°C. Based on the sequence of the PCR product a new probe was constructed, completely homologoustothepypgene inEhalophila. Thisprobewas used toisolateapositive clone (pYAMA18) byscreeningamini library of 2.4 kb PstI chromosomal fragments from Ehalophila in phage M13mpl8. A950 bpPvuII fragment from pYAMA18, containing the

pyp ORF,wassubclonedinMl3mpl8togive pYAMA958.

Cloning of theRs.salexigens pyp gene

The probe usedto clone thepyp gene from Ehalophila was used in

heterologous Southern hybridization experiments with Rs.salexigens

chromosomal digests.A mini library, containing sized PvuI-SalI

frag-ments in phage M13wasscreenedbyhybridization withthe same probe,

leadingtothe identificationoftwopositiveclones. A 1.4kbfragment containing the pyp gene was made blunt by Klenow treatment and reinserted into theSmaI linearized phageM13mpl9, yielding pS16.

Sequencing

Both strands of the 1.8 kb Ehalophila PvuII-PstI fragment and the Rs.salexigens1.4 kbPvuI-SalIfragmentweresequencedusing universal andgene-specific oligonucleotides; thesequence strategies areindicated in Figures 1 A and 2A. Sequence information was obtained by the

dideoxy chain terminationmethod(Sangeretal., 1977),using[35S]dATP

and a modified T7 DNA polymerase sequencing kit (Sequenase: US

BiochemicalCorporation, Cleveland, OH), aswell as through the use

of fluorescently labeled dideoxy nucleotides and a thermostable Taq polymerase withtheDyedeoxy terminator cycle sequencing kit (Applied Biosystems,FosterCity).

Identification of theR.sphaeroidespyp gene

Chromosomal DNA fromR.sphaeroides2.4.1 was used as templatein aPCRusing 10cyclesofannealingfor 1 minat25°Cand 25cyclesat

35°C. Denaturationandelongation wereperformedin all35cycles for

1 min at 95°C and 72°C respectively. Primers were based on known pyp sequencesand restriction sitesBamHIandHindlll (underlined)were

introduced to enable directional cloning:

GCGGATCCGCCTTCGGC-GCCATCCAGCTCGAC(NTPYPI)and GCGCAAGCTTCTAGACGC-GCTTGACGAAGACCC (CTPYP1). The PCR product obtained was isolated from agarose gel and inserted into phagesM13mpl8/19. Both strandsofthe PCR product were sequenced.Hybridization of the PCR product with R.sphaeroides chromosomal DNA was performed as

described(Engler-Blumetal., 1993).

Identification of the chromophore of Rs.salexigens PYP

A colorless Rs.salexigens culture, grown aerobically in the dark in Hutnermodifiedmedium as described(Hoffetal., 1994b),wasdiluted

twice in the same medium and incubated anaerobically at 42°C in a

Xanthopsins:genes andoverexpression

tungsten lightbulbs, yieldingared culture after 96h.The solublecell

fraction of500 mlofaerobicallyand anaerobicallygrowncultureswas

prepared as described (Hoffet al.. 1994b). Proteins were precipitated

with 10%(v/v)trichloro-acetic acid and washedoncewithdemineralized

water. Pellets were resuspended in 5 ml demineralized water and incubatedovernightatpH 12(leadingto acompletesolublization of the

proteins) to hydrolyze thiol ester bonds, followed by acidification to

pH4 withhydrochloricacid andaceticacidtoneutralize thechromophore

for optimal extraction. Before extraction, protein concentrations were

determined with the Bio-Radproteinassay,asdescribedbythe

manufac-turer.Chromophore extractions were performedby mixing thoroughly

with 15 mlethyl acetate.followed by5 minofcentrifugationat 120 g. The organic phase was washed twice with 5 ml demineralized water

and dried by air. To substantiate the result ofour analysis, the same

chromophore extraction procedure was carried out using the purified

Rs.salexigensPYP(Meyeretal.,1990).Air-driedsamplesweredissolved indistilledwaterandinjectedina50 tmfused silicacapillaryTSP050375

(CompositeMetal ServicesLTD)withaninjectiontime of 0.2 min and injectionpressureof40mbar. Thesamplewasanalyzedin 60 mM Tris/

30mM valeric acidpH8.2,throughacapillarywithaneffectivelength

of 55 cm,at25 kVand -12 tA.On-columndetection was performed

at 284 nm (determined as the wavelength at which transp-coumaric

acid maximally absorbs in theTris/valeric acidbuffer), with a UVIS 200 detector (Linear. Fremont). As areference tranis p-coumaric acid (Sigma. St Louis, MO) was used. To confirm this identification,

p-coumaric acid wasalso subjectedtoelectrophoresis in25 mM borax

buffer, pH 9.0 at 25 kV and -35 tA. The amount of detected trans

p-coumaric acid was calculated from thepeak area usingthe software

Caesar for Windows (version 4.02. 1990, Prince Technologies). As a

reference, 11.0nI of tratlsp-coumaric acid(Sigma)was injectedin the concentration range from2.5 to75 tM. showingalinear relationtothe detectedpeakareas.

Construction ofoverexpression plasmidsand

overproductionstrains

A 0.45 kbAvall fragment from pYAMA958,containing thep!p ORF from Ehalophila. was ligated into theSmnaI-linearized overexpression

plasmid pT713 (Studier et al.. 1990) to yield pTY13. which was

transformed toEcoli BL21. Overexpressionin pT713 is basedon the

strong viral T7 promoter 10. The gene coding for the viral RNA polymeraseislocatedonthechromosomeofEcoliBL21.downstream ofaninduciblelac promoter(Studieretal., 1990).

Aconjugativebroadhost rangeoverexpressionsystemwasconstructed by ligating the0.45 kbAvail fragment. describedabove, intothe Pstl

polylinker site of pCHB500. pCHB500 is a broad host range vector, containing two promoters directly upstream of the polylinker site: the

E.coli Plac promoter and the Pcyc promoter that supports anaerobic expressionof thecvcAgenefromR.capsulatus (BennigandSommerville,

1992).Theresulting plasmid pART3wastransformed intothe conjugative strain Ecoli S17 and then transferred toR.sphaeroides DD13 (Jones etal., 1992)byconjugationonLBagarplatesfor 4.5 h.Transconjugants were selected on LB plates containing tetracyclin (10 tg/ml), strepto-mycin (5 .g/ml) and kanamycin(20 tg/ml). Thetransconjugants were

subsequentlygrowninliquidmedium undersemi-anaerobicconditions,

allowing pigment synthesis.

Athirdoverexpressionsystem involvedtheheterologous

overproduc-tion ofan affinity-tagged version of PYP from Ehalophila in Ecoli.

Theexpressionvectorwasconstructedbydirectional insertionofaPCR productinto the expressionplasmid pQE30 (Qiagen, Hilden). ThePCR

product was obtained using pYAMA18 as template ina reaction with the primers

GCGGATCCGATGACGATGACAAAATGGAACACGTA-GCCTTCGG (NTPYP2), containing the BarnHI site (underlined) and

CTPYPI (see above). Use of NTPYP2 results in the presence ofan

enterokinasesiteinthe recombinantprotein,allowing proteolyticremoval of the affinitytag. This tagisformed bysix Hisresidues,encoded by

pQE30(Qiagen).ThePCRwasperformed usinganannealingtemperature of 60°C for 30 s and extension at 70°C for 30 s in 30 cycles. The

resulting PCR product was digested with BarnHI and HindIll, ligated intopQE30(Qiagen)toyield pHispandtransformedtoEcoliM15.The

colonies, resistant against ampicillin (100 ,ug/ml) and kanamycin

(25

tg/ml),

were shownto contain theconstructby colonyPCR, using thetwoprimersdescribed above.

SDS-PAGE, Western blottingand RIEP

SDS-PAGE was performedin aBio-Radmini slab gel apparatus

(Bio-Rad, Hercules, CA) according to Laemmli (1970) as modified by

Schagger and Jagow (1987) for improvement of resolution in the

5-20 kDa range. Gelswerestained withCoomassie brilliant blue G250. Western blotting and immunodecoration were performed as described previously(Towbin etal.. 1979; Hoff etal., 1994b). RIEP was carried out asdescribed(Hoffetal.. 1994b).

Heterologous expressionof PYP

E.coliBL21/pTY13 and EcoliM15/pHisp were induced to express the

heterologousgenebytheaddition of I mM IPTGtowell-aerated cultures ofexponentially growing cells at anOD660of 1. Cells were grown at

37°C in well-shaken Erlenmeyers, or in a well-aerated 10 I fermentor (New Brunswick Scientific, New Brunswick). Production of PYP in R.sphaeroides was inducedby growing theorganismsemi-anaerobically

in two-thirds filled, slowly shaking Erlenmeyers, using Luria Bertani broth withappropriate antibiotics. The resultingEcoli andR.sphaeroides

cells were sonified three times for I min while cooled on ice, and centrifuged at 200 000 g for 3 h at4°C toobtaina clearsupernatant containing the overexpressed product. Absorbance spectra of these fractions were measured with an Aminco DW2000spectrophotometer

(SLM Instruments). In addition, these fractions were used for SDS-PAGE, Western blotting and RIEP analysis, as described above.

Isolation andcleavage by enterokinase of histidine-tagged PYP

Ultracentrifugation supernatants from Ecoli M15/pHisp, induced with IPTG, were incubated with Ni-NTA resin for 1 h at 4°C, as described

by the manufacturer(Qiagen). The resin waspacked in a column and eluted, either by an imidazole gradient orby a pH gradient, as described by the manufacturer. The protein elution pattern was analyzed by measuringthe absorbance of theeluting fractions at 280 nm. Cleavage

ofhistidine-tagged apoPYP was performed at37°C for 5-24 h usingan

enterokinase:PYP ratio of1:50(w/w).

Reconstitution of holoPYP

Reconstitution of theheterologously produced apoPYP was achievedby

addition ofthep-coumaric anhydride,dissolved indimethylformamide

(DMF),asdescribed forthereconstitution ofthe apoPYP,obtainedfrom

E.halophila (Imamoto et al., 1995). The p-coumaric anhydride was

synthesizedasdescribed(Imamotoetal., 1995).

Massspectrometry

The integrity of histidine-tagged apoPYP and reconstituted

histidine-taggedholoPYPwasverifiedby electrospraymassspectrometry(ESMS).

Typically,20pmolofproteinwasdissolved in 10 ml CH3CN:water:formic acid(1:0.9:0.1; v/v) andinjected intotheelectrospraysource of a VG

Bio-Qmassspectrometer(VG Organic, Altrincham,UK) at aflow rate of 6ml/min,deliveredbyaHarvardSyringe Pump 11 (Harvard,South

Natick. Ma). Nine-second scans, covering the 650-1550 amu range,

were accumulated during 2.5 min. The spectra were collected and

processedusingthemasslynx software providedwith the instrument.

Acknowledgements

The authorsareverygratefultoR.Kok and J.van Thor for their advice

on the use of molecular genetic techniques and to J.van Dijk and

W.Spijkerforcloning theRs.salexigenspxpgene. Wethank X.Xu and H.Vonk for performing the capillary electrophoresis experiments and

H.P.M.Fennemafor synthesisofthep-coumaric anhydride. We would liketothank B.Poolmanforhishelpwith theinitialPCRexperiments.

Weareverythankful toT.E.Meyerforsupplying information concerning

oligonucleotides YS- I and YS-2 and the purified Rs.salexigens PYP. W.D.Hoffwassupportedbythe NetherlandsOrganizationfor Scientific

Research(NWO) via the Foundation forBiologicalResearch (BION). J.Van Beeumen is indebted to the National Fund for Joint Basic Research forfinancial support(Contract 32001891).

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Note added in

proof

Recent resultscastdoubton ourstrainassignmentinE.halophila.