Sequence and functional analysis of an Escherichia coli DNA fragment able to complement pqqE and pqqF mutants from Methylobacterium organophilum

( 1996) 78, 822-83 1 0 Soci@tGfranqaise de biochimie et biologie mol&ulaire / Elsevier. Paris

Bio~ hintie

E Turlin, F Gasser, F U&PRt@dutim

de I' Expressior; Gfnhticpre, D~partctw~t de Bioc*himie et GPnPtique Molkuluire. lnstitlrt Pasteur, 28, rue hr Dr-Rmrx, 7.~5724Paris cedes 1.5, Fruncx~

(Received 20 May 1996; accepted 27 September 1996) Summary - A 736 I kb fragment of E wli chromosomal DNA able to complement ~lqc/E and !qqF mutants of Merhvlohuc,tcr.ilrnl orgumhas been sequenced. Five open reading frames (ORF) have been identified. Four ORFs (102, 103, 106 and 107), belong to a single transcription unit. They are separated by a transcription termination site from a fifth ORF (ORF109). Polypeptides of 28, 85 and 82 kDa encoded by ORFs 102, 103 and 106 respectively were visualised in maxi-cell experiments. Both ORF106 and 0RF107 are required for complementations of PqqE andpqyF mutants from M orpnmphilum. The polypeptides encoded by ORFs 102, 103 and 107 have no homologies with the producls of pqq genes previously sequenced from Acirletohacter t*akoaceticws, Klehsiella pneunwniue, and Merh~Mmc~ter irm e_rtcwquens AM 1. The polypeptide encoded by ORF 106 shows homology with the pqqF gene product of K lmeunroniae, and seems to belong to a family of zmc proteases. The sequence of 0RF109 is identical to the sequence of the ,qadEI gene of E coli encoding for a glutamate decarboxylase.

philum

pyrroloquinoline

quinone / nucleotide sequence analysis I chromosomal insertion mutants / E co& / Methylobacteriurn organophilurn

Introduction

Pyrroloquinoline quinone (PQQ) is the cofactor of the quinoprotein bacterial dehydrogenases. Most of these enzymes are located in the periplasm or in the cytoplasmic membrane ofGram-negative bacteria [ I 1. PQQ is synthesized from two precursors, tyrosine and glutamic acid [2). The different enzymatic steps between these precursors and PQQ have been hypothesised 13J but no intermediary product has been identified yet. Processing of PQQ, ie how PQQ is transported through the cytoplasmic membrane and assembled with its apoenzyme is also poorly understood [4]. After complementation analysis, six distinct ~44 genes were found in M or;panophilum [5] and seven in M e-t-twquens AM1 16). Correlation between pqq genes of these two methylotrophs was established [6]. In M estorquens, three other genes, moxA, moxK and mo,uL (formerly mo_vAI A2 AS) [7] were shown to be involved in the association between PQQ and the methanol dehydrogenase apoenzyme [8, 91. In A calcoaceticw, and in K pneumoniae, genes corresponding to the first cluster of methylotrophs ~44 genes (ie pqqABCGD) are also present. In A calcoaceticus, pqq genes

*Correspondence

and reprints

in the order III, II, I, V, IV were mapped in a 508%kb DNA fragment and sequenced [ 109. In K pwunw~~iae six contiguous IX/~ genes (pqqA to pc/c$) were mapped in a 6940kb DNA fragment and sequenced [ 1 l].The pqqCGD gene products of M ~J,WW~~WW were shown to be strongly homologous to those encoded respectively by genes I, V, IV of A (‘NI~‘OiI(‘CJti(*ll,s and genes IX&, B, A of K pwumoniae 16, 10, 1 I]. Data bank comparisons did not provide any information on the nature and on the role of the polypeptides encoded by pyy genes of M extorquens AM 1 [6] and A calcoaceticus [lo]. In Kpneumoniae, the 84 kDa protein encoded by ~49F shows homology with a family of prokaryotic and eukaryotic zinc proteases [ 1 I]. It was then concluded that a protease activity was necessary for the PQQ biosynthesis pathway, and the gene product of pqc-A, a 23-amino acid peptide, gene was proposed as a template for PQQ biosynthesis [ 111. According to this hypothetical model the matrix provides the precursors of PQQ glutamate and tyrosine. After ring closure hydroxylation and oxidation, the molecule of PQQ is excised by the gene product of ~94F [ 111. In several experiments, the cloned ~44 genes were tested for PQQ production in E coli wild type because this organism does not synthesise PQQ although containing a constitutive GDH apoquinoprotein that can be activated by added PQQ [ 121. Thus, the four yqq genes III, II, I, IV from A calcoaceiicus were claimed to be sufficient for PQQ biosynthesis because their expression in a strain of E cnii Apts produced colonies acidifying glucose-McConkey agar as a

gous to pycj gene

a single gene from Pseudomonas cepwicr was shown to be sufficient to trigger PQQ production in E cwli. [ 161. This gene has no homology with the previously sequenced lx/cl genes [ 16]. These conflicting data regarding the number of ~xly genes necessary for PQQ biosynthesis have to be re-examined in the light of another work showing that, in E wli, a single spontaneous mutation allows PQQ production in detectable amounts [ 171. The E c*oli PQQ+ mutants (EF strains) were obtained from E coli Aprs, as clones able to grow in glucose minimal medium. In membrane pre tions of EF strains, GDH activity was evidenced and was assayed [17]. Thus, in E cdi wild type, genes required for PQQ production are present but they are not all expressed. A gene library of chromosomal DNA from E mli was built in the cosmid vector pLA29 17 and tested for corn-pletnentation of M nr-,qurwphilrm mutants impaired in PQQ biosynthesis [ 1S]. A cosmid able to complement both IX/L/E and /x/c/F mutants of M or:~crnc)l?hilrrnI was selected and the genes involved in these complementations were located in a 736 1-kb DNA fragment [ IS]. In this report we present ~he complete nucleotide sequence of this DNA fragment. its localisation on the chromosome of E rwli and the visualisation of the encoded proteins. The roles of ORFs, in corn--” p 1em e n t at i on s o 1‘/x/(/E a n d IX/# m u t a II t s o 1’ M or;cCanc~~~trillrnl, were also investigated.

aterials and methods

Transformations wcrc monitored uhiilg the calcik.inl dtlloi I& method [ 3 1 I Iysatcs and transductions WI-e carried out as dcscribed by er 1291. Bacterial matings with E coli strain 517.1 containing pLA27 17 derivatives as donors and M of.~trffr)l~ifilirrwr as recipient were performed as previously described 1.91.TnS inscr(ions were performed using the defective h-l67 [ 211. Cof~.stf~irc~riofi0f’c.hf~of~fo.soff 1~1if f.wfTioff ffffrttrffrs

Resistance genes from p P AX2 [2X] derivative\ were clol,cd into restriction sites of different open rczdin g fran1tJs. DNA f‘rap1cnt, containing these open reading frames disrupted by 111~resistance (Tene(Ap or Cm) were isolated using electroclution and introduced Ky transformation in a IIVD deficient strain (32j.

Large-scale DNA isolations were carried out using Pltlxnlid Maxi Kit (Qiagen Inc) as recommended by the manufactutcr. Small->c:ilc DNA preparations were performed according to Birnboim ~lnd Doly 1331. Endonucleuse digestions. alkaline pho$luta~ trcatmerits. and ligations were performed as described by Maniatis (11 trl (31 J. Overlapping clones for DNA squencing wc‘rc built uhing Cyclone 1 Biosystem from IBI (%I. Gaps were filled using syr?thetic primers.

Scyucncing reactions were pcrfiWnied in microtitration platc~ with a ~11ple charger f‘rom Gilson (model 222 plus dilutor 301 ) using the didcohy method I.351with the’T7 poIymc‘r;t~c (Pharmaci;l). The DNA sequence reported in the manuscript i\ dcpositccl i:~ rllc EMBL Genbanl; under the accession number X7 19 17. lhtti

lftif

ftllif f,q

Each sequence gel was read using a digitali/cr. The squcnces wcrc overlapped usin, (1sofiwarc written by Staden 1Ml. Squtmce corn parisons were done using BLAST I37 1.

The bacterial strains, phages and plasmids used in this study are listed in table I.

The restriction map of the 7.6kbp DNA fragment was compared with the whole E wli restriction map of Kohara VI trl 1381 using software of Mediguc ct (111391.

E co/i strains were grown on LB medium [ 291 or on Mac Lennan 1301 minimal medium supplemented with vitamin Bl and with 0.4% glucose or gluconate as carbon sources. M of~~~crffo~~~hilfffff strains were grown on Mac Lennan minimal medium supplemented with 0.2% succinate or 0.5% methanol as carbon source. When required, filter-sterilised PQQ (Fluka) was added at a final concentration of 1 nM for E coli strains or I FM for M of;paffofphilrrm strains. Antibiotics were added at a final concentration (pg/mL): ampicillin (Ap), 50; chloramphenicol (Cm), 40; kanamycin (Km),

Analysis of plasmid-encoded proteins u’as performed in LIY(
824 ____ ________ Table I. Bacterial strains, phages lnd plasmids. - Straills ____

FB8 FB8 Apts FB8 Apts galP EF260 v355 s17.1 SE 5000 TGl Meth_vlohacterium nr;panophilum

~_____.___

..~. __

___Rcj __~_._ __~~__._____________

_ ___-

--~~ Chcrr-crc~trr-istic.s __________ -.

F; wild type F A (ptsH pts1 W) F A @tsH ptsI c’rr) ,qalP::TnZO F A (~JYHptsIc1~) galP::TnlO able to produce PQQ c, lac - 3350, ,qcrlK2, gal T22, h-, WL’D1014, qx L179, IN (wzD-~rlE) 1 IWA thi pro, hsdR, chromosomal RP4-2TnI::ISR 1,Tc:: Mu, Km::Tn7 F, araD139, A (~WRF-lac) U 169, rpsL150, ~.eIAl,jlh B5301, de&l, ptsF25, rhsR, recA56 lac -polo AB thi .\vpE hsd A5, F’ tra D36, proAB+ ltr& 1rcZ AM 15 DSM 760 (wild type) pqqE mutant pqqF mutant

430 530

A467

-._ ----____

19 17 17 17 20 21 22 23 Deutsche Sammlung von Mikroorganismen 5 5

h22!, (*I857. IYS:: TnS, 029, P80 a

24

IncP, Tet’, Kanr

25 26 26 27 27 Pbarmacia (cal no 274987-0 I ) 28 18 1X 18 1X This report This report This reporl This report

Plasmids PLa 2917 pTZ18 pTZl9 M13mp18 Mi3mp19 pUC4K pHP45S2 pBGT3 pBGT4

pBGT5 pBGTb pBGT7 pBGTX pBGT9 pBGT 10 pBGT30 pBGT40 pBGT322 pBGT369 pBGT300 pBGT303

Tct’ Tet’ Tet’ Tel’ pBGT3 derivative {leleted for IWO bp Ystl fragment pBGT3 derivative ‘deleted for 700 bp Pstl fragment 2762 bp S,qlll fragmerat from pBGT3 cloned in pLA29 17 pBGT3 derivative de’feted for 150 bp Hind111 fragment pBGT3 derivative cctntaining the Amp’cartridge from pKT254&2- Ap inserted in ORF 106 pBGT3 derivative containing the Amp’cartridge from pKT254&2-,ap inserted in ORF 103 pBGT3 derivative cont‘tining a Tn5 insertion in ORF 109 pBGT3 derivative cont,&ing a Tn5 insertion in 0RF107 pTZ 19 derivative containing z 4 138 bp HirldIII fragment from pBGT3 pTZ 19 derivative containing a 2762 bp B,qliI fragment from pBGT3

Results and discussion Nucfeotide sequence of the E coli DNA jkagmetzr contained in pBGT3 As described in a previous paper, pBGT3, a derivative of pLA2917 [25], containing a 736 1-bp fragment of E coli

This report This report This This This This

report report report report

DNA was able to complement 1749E and p99F mutants of ‘Mot-,patmphilurtt [ i 81. Various restriction fragments from pBGT3 were subcloned in M 13mp 18 or M 13mp19 and overlapping fragments of each subclone were built and secl’lenced as described in Materials and methods. The compl,ete nucleotide sequence (fig 1) was comprised of 7361 bp anId showed a 50% G+C content. The five ORFs identified

825

The restriction map deduced from the sequence was used to enced 7361 kb frag ] using the softwa al [39]. The fragment was unambiguously tion 1588.50 kbp (33.65 near tetC the terminus ping since the ,?a( gene was shown to be located at 1588 kbp on the E cA’ NA chromosome [ 4 11.

M L V S-N GATCAATCTGGGkGACTGATGAAATCGCiZX&XxcATTT~CTGGTATCGAACAATTT

N

L

ORF 102 CO

PEKISIHGASPYQNAYL IDG CC~GAGAAMTCTCTATTCACGG~GCGTCGCCCTACCAGAATGCCTATTTGATTGACGGT

:140

120

IbkTNNLNPANESDASSATN ATTAGTGCAACTAATAACCTGAACCCAGCGAATGAGTCCGATGCCAGTAG'?GC&%XF&T

1200

180

ISGMSQGYYLDVSLLDNVTL ATTAGCGGGATGTCACAGGGGTATTATCTTGATGTcAkTTACTGGACAATG~CGCT"T

1260

240

YDSFVPVEFGRFNGGVIDAK TAn;ACAGn"m;TGCCGGm~l~~TCGfTTCAA1Y

132t

300

IKRFNADDSKVKLGYRTTRS ATCAAACGCTTCAACCCTGATGATAGCAAGGT~?.AATTGGG'M'ATCGCACTACGCGTTCG

1380

360

DWLTSHIDENNKSAFNQGSS GACTGGTTAACATCGCATATCGATGAG~TAACAAGAGCGA

1440

420

G S T Y Y S P D F K I( N F Y TLSFNQ GGAAGTACTTATTACTCCCCAClTTTTAAAAAGAACTTTTATACCTTGTCGTCGT'PTAATCAG

1500

4HO

ELADNFGVTAGLsRRQSDIT GAACTCGCTGATAAC~CGCG~ACCGCCGGTTTATCGCGCCGCCAG~~ATATCACC

15bU

541)

R A D Y V SNDGIVAGRAQYKNV CGCGCGGATTAn;TTTCGAATGACG~CATTGTCGCCGGTCG~ACAGTAT~CGTT

1620

ILSSGEKQRIALARLILRRP TATTCTTTCCAGCGGCGAAAAACMCGTATCGCCCTGGCACGATTMTTTTACGACGTCC

600

IDTALSKFYLVCQRPLYPRL ATCGATACTGCATTAAGCAAAAATTTTACCTGGTTTG‘XAGCGACCGCTTTACCCACGATTA

rGE0

KWIFLDETTSHLEEQEAIRL GhAA~ATATTTCTn;ACGAAACTACCTACCTCTCATCTTGA~~CAAGAGGCTATCCGCTT

660

T L K Y T G S S R D "NTSTFPQSD ACCTTAAAATATACCGGCTCCAGCCGTGATTATAATACCAGCACC~CCC~AGTC~AT

1740

IMVTHQP LRLVREKLPTSGV ACn;CGTTTAGTGCGTGI\ARAACTACCCACAAGCGGCGGCGTCATTA~TTACACATC~CC

720

REMGNKSYGLAWDMDTQLAW CGCGAAATGGGTAATAAATCCTATGGTcTGGCATGGGATATGGATACCCAGCTCGCAT%

1800

S A V L ?? GVWNLADDICDI CGGTGTCTGGAACCTGGCCGATGATATTTGTGACATTAGCGCGGTTTTATAAAAACAATA

780

SDYTRHDH AKLRTTVGWDHI GCCAAACn;CGTACCACCGTTGGTTGGGATtATATTAGn;CAT

18GO

AAAGCCCTGCTGTACGGAGATATAAATAAATGAAGCGAGTTCTTATTCCTGGCGTCATTT

840

DIWYTELSCTYGDITGRCTR GACATCTGGTACACCGAACTTTCAl'ZTACATATGGTGATATTACAGGGCGT~CACccGT

1920

G G L 0 H ISQAVDNYTFKTRLD GGCGGATTAGGACACATTTCCCAGGCTGTAGATAATTACACCTTCAAAACAcGccTGGAc

1980

WQKFAVGNVSHQPYFGAEYI ~;GC~TT~GCCGTGGGTAATGTTTCGCATCAACCCTACTTCGGCGCGG~TACATC

2040

SWFIYKYDELAELAAVIDRL AAGCTGGTTTAT'ITATAAATATGACGAACTTGCTGAACTGGCTGCGGTTATCGATCGCTT Y E F H Q LT E Q R P T N K P KNC Q GTATGAGTTCCATCAACTCACTCAACAGCGCCCTACGAATMGCCTAAAAATTGCCAACA

H

AVQVADAS IRTPDNKIILEN TGCGGTACAAGn;GClXATGCGAGTATTCGTACGCCTGATAATAAGATCATiiTTAGAGAALNFHVSPGKWLLLKGYSGAG CCTGAACTTTCA'IG'ITTCGCCAGGCAAATGGCTATTACTGAUGGCTACTCTGGCGCGGG KTTLLKTLSHCWPWFKGDIS AAAAACCACACTGCTTAAkACATTATCCCACTGCTGGCCG.~TTTAAAGGTCATATTTC SPADSWYVSQTPLIKTGLLK TTCTCCTGCTGACAGTTGGTATGTGTCACAAACACCGTTAATCUAACCCXCTTACTGAA E I I C KA L p L PV D D K AGAGATTATTn;TAN\GCACTTCCCCC~CCCGTAGATGTACT

5

L

S

E

V

IHDHDRWGD HQVGLGKLAAR GCATCAGGTTGGTCTTGGGAAnTn;GCTGCtiCGTATTCA~ACCACGAT~GCTGGGGAGA

MTKNMYMHFF TATGTGGCGCTGATGTGGCwCCGTCGmACTATTT PVNGNTHYT EEMTVYAPVPV GRAGAGAn;ACGGTCTATGCTCCn;TCCCn;TACCCGTAAATTA~ACC

L

ORF 103 9'30

960

SESIERLPTGNGNISDLLRT AGTGAAAGCATCGAGCGTTTACCGACCGGGAATGGCAATATCAGCGATCTGCTGAGAACC

1020

YSDAWTERHNQSESYVINAA TATTCCGATGCGTGGACTGAACGCCATRACCAGTCTGAATCCTA%TGATTAA=cT=c

2100

NPAVRMDSTQSTSLNQGDIR AACCCn;CGGTACGCAn;GATTCAACGCAAAGTACCTCGTTG~CCA~GAGAT~TTCGC

1080

IYHK6KGRLGIDN GKKTNHT C+GAAAGAAAACTAACCATACCATTTACCATAAAGGTAAAGGCCGCCTGGGAAT%AC~C

2160

Fig 1. Nucleotidic sequence of the 7361 bp HindIII-Ec*cjRI DNA fragment of pBGT3. The predicted amino acid sequences of the different open reading frames are indicated as follows (position): QRF102 (42-772), ORF 103 (87 l-3 174). ORF 106 (323 l-5375), ORF 107 (5204-6013), and ORF 109 (63757361). The putative promotor boxes, terminator, ribosome binding sites and the ATG translation start sites are underlined.

M R N GGAT~'TTACTTAAC~GGGTAAATTCGCCCGGTTTTCGCAT?.?QAQAT~TTA#&AGAAACC

826 YTLYNADRISWRNVSLMPSV TCGCATTAGCTGGCG TAATGTGTCATTAATGCCCGGCGTG TACACGCTGTATARYDYDNYLSNHNISPRFHTE CGiTATGAcTATGACUCTA TCTGXMAccACAATATcTCCCCGCG~ATGACGGAA WDIFANQTSMITTGYNRYYG TGGGATATTTMGCTAATCAAACCTCAATGATTACGACAGGTTATAAcC~ACTA~C RNSWTESVS GCTGGACGGUTCGGTAXA

GNILDMGLRDI GGGAATATTCTTGATA~~A~TAl%GCAATA

2220

LCFLL_TLVATLLLPGRLI.AA TiXGTTTcTTACTGACGTl'AGlGGCAACTC'CGTTGCTCCCCGGGCGGC~A~GcCGCCG

3309

2280

ALPQDEKLITGQLDNGLRYM CCTTACCGCAGGATGAAAAAAAAGTTRAATTACCGGGCAACTGGACAATGGCT~CGATATATGA

3360

2340

IYPHAHPKDQVNLWLQIHTG TITATCC~A~TCATCCAAAGGATCAGGTAAA'l"rPAT

3420

2400

SLQEEDNELGVAHFVEHMMF CATPCCAGGAAGARGAC~~~~~G~~CArA

3480

2460

NGTKTWPGNKVIETFESMGL ACGGCAC~cA'XGCCGGGTPATAAAGTCATcGAAACA~AGTCAATGGGcCTGC

3540

2520

RFGRDVNAYTSYDETVYQVS GTTKGGXGCGATGTTAATGcCTATACCAGcTATGACGAAACGGTGTATCAGG'i'GAGTT

3600

2580

LPTTQKQNLQQVMtiI F S TGCCGACTACGCAGAAACATGCAACAAGTGATGGc~TCTTCAG~~GAGTA

3660

2640

NAATFEKLEVDAERGVITEE ATGCCGCAACCTl%ZdAAACTCGAAGTAGACGCTGAACGCn;AAT

3720

2700

WRAHQDAKWRTSQARRPFLL GGCGlWXCATCAGGAlGCGAAATGGCGCACCTcTCAGGCGcGcCGCCCT~CCTGC~

3780

2760

ANTRNLDREPIGLMDTVATV CAAATACCCGTAATTTAGACCG'XPACCTATCGGCCTGA!IGGATACTGTCGCCACGGTCA

3840

2820

TPEQLRQFYQRWYQPNNMTF CACCGGC 4CAATTGCL~XAATTTTATCAACGCTGGTATCAPSCAAATAATA'l'GACCT~A

3900

3960

GNKNSDALSGFENDELAMGL TmGAAAAcGAlw+A~AA~TrG

C QQICIGANVIARANYVYREAL CAOCAGlUMTCGGTMWUCGT VQISICSSRTDSATKTTITEY GTACAM-GmGTCGT

~AAXTA'X'MTACCGTGAAGCGCTA

ACCGACAGCGCGACTAAAACCACCATTACTGAATAT

NNDGKTKTHSFSLSFELAEP MCMCGATGGCAMACCAAAACGCATl'CGT'XAGCcTcAG

mTTGAAc~cGAAccc

LLYRQVDINPQIVFSYIKSK CTGCTATACCGCCAGGTAGATAlTMCCCACAAATTGTCTTTAGCTATATcAAGAGcAAG

YNGNLVSYDSVPVADFNNPL TTACGATAGCGlTCXAGTGGCAGATTR'AATAACCCCATTA TAT&AC

QRF 106 3240

EWS

2880

K DN L S IVIGDIDSKEALALI TCGTGGTCGGCGATATCP~CAGTAAAGAAGCGCTC4XGCTGATAAAGGATAAT-l'T~GTA

ILGKTNAQY LAWQEARKARI CTGGCCTGGCAAGAAGCGCGTAAAGcTCGcAlTATCCTTGGTAAGAC~TGcGCAATAC

2940

PTKAENH K L P AN KAA ENRVW AGCTTCCbGCTAACAAAiiCAGCTGUAATCGCGTCTGGCCGACAAAAGcCG~CCACC

4020

ISEYSDYKQYVDEKLDSSLT ATCAGcGAATATTCAGATTACARGCAGTATGmACGAAAAAC

3000

L R F N I INDKENRVNGIALYY TGCGCTTTAATATCATTAATGATAAAGAAAACCGGGTGA&CGGCATCGCACTCTATT.'.TC

4080

WDTRLSWTPQFLQQQNLTIS 'l%GACACCCGCTTGTCCTACGCCAC~TTTCTGc~C~C~CCTGACGATCAGT

3060

I E Q A E.lr; S RLPMVQVNDEQSF GCCTGCCAATGGTACAAGl'GAACGATGAGcAAAGCTTTATCCX#.CAFiGCTG~TGGAGCA

4140

A'DILNVLDSKTAV-DTTNTGV GCCGATATTCTCAATGTACTGGATAGCAAAACCGcTCiTTGATACAACGPACACCGG'TGTG

3120

NLVQ L F N Q R LQ E R I Q S G ELK TiiTTAGTTCAGCTfXTCAATCAACGTCTGCAGGAACGCATACAGTCGGGCGAGTTGAAGA

4200

3180

TISGGTARSVKIAPDYQSLF CTATTTCTGGCGGCACTGCGCGCAGCGTTAiiU~CACCCGATTATCAGTCGCTGTTTT

4260

ADDRTKLLQENQIAQFTAAD MLMTARNYCKKIRLHSLLPQ TGCTGATGACCGCACGAAATTACTG~AAGAAAATCAGATTGCACAGTTTACTGCCGCAGA

5280

ALAADRQLFSSPADITFVIV MRWLPIANCFHLQRISRLSL TGCGCTGGCTGCCGATCGCCAATTGTTTTCATCTCCAGCGGATATCACGTTTGTcATTGT

5340

4500

GNVAEDKLVALITRYLGSIK SVMSQKTNSWR* CGGTAATGTCGCAGAAGACNCTCGTGGCGTTAACPTAGGATCAATCAA

5400

4560

H SDS P LAAGK PLT RATDNAS ACACTCTGATTCGCCATTAGCCGCAGGTAUCCATTAACTCGCGCGACGGACAACGCATC

5460

4620

v T VKEQNEPVAQVSQWKRYD GGTTACTGTAAAAGAACACCTGTGGCACAGGTYI'CACAGTGGAAGcGTTATfZ

5520

4680

SRTPVNLPTRHALDAFNVAL Tl'CCCGGACACCTGTTAATCTGCCGACGCGTATGGcGcTcGATGCTTTTAACGTcGCACT

5580

4140

AKDLRVNIREQASGAYSVSS GGCAUAGATCTACGTG'M'AATA'ITcGTGAACAGGcATC?GGAGcATACAGcGTTTc'iTC

5640

4800

RLSVDPQAKDISHLLAFTCQ TCGCCTCTCGG'PTGATCCTCAGGcCAAAGATATCAGTcATTTGCTGGCTTTTACTl'GTCA

5700

4860

PERHDELLTLANEVMVKRLA ACCAGAACGACATGATGAACTGTTAACGTTAGCGAATGAAGTGATGGTTAAGCGTCTGGC

5760

4920

KGISEQELNEYQQNVQRSVD TAAAGGGATCAGTGAGCATG~TGAATACCAGCAAAACGTTCAGCGCAGCGTCGA

5820

4980

IQQRSVQQLANTIVNSLIQY TATCCAACAGCGTAGCGTTCAACAATTAGCGAACACTATTGTAAATAGTCTTATTC~TA

5880

5040

DDPAAWTEQEQLLKQMTVEN TGACGATCCTGCAGCATGGACTGAGCAGGAGCAATTGTTGA&iCA&4TGACGGTAGAGAA

5940

5100

VNTAVKQYLSHPVNTYTGVL TGTTAACACTGCCGTTiCAATATcTTTCTCATCCGGTAAATACTT,ATACCGGAGTATT

6000

5160

L P K ?? ATTGfrAAU~MTAACAG

6060

KISLNMDFTHQPSGLVWANT MGA~CTTAAACATGGATTPCACGCATCAACCGAGCGCG

ATYASGRTFWLDVSMKF. GCGACCTACGCCAGTGGCCGTACTTTCTGGCTTGATGTcAGcATGA4ATTTTAATAGTTC

4320

4380 RLTWLKNAVPQQAERDLRHL GCCTCACCTGGCTGAAAAA TGCGGTTGATCAGCMGCTGTGATTTACGTATGC~

TSRLASSSLNNTPFLSPEET CCAGTCGC~A~CA~ATTAAATAATACGCCGTTCTTGTCGCCGGAAGAGACAT YQLSKRLWQQITVQSLAEKW A?rM~CGTCnACCG~~G~~~~

4440

QQLRKNQDAFWEQNVNNEVA AGCAGTTAAQAAAGAACCAGGACGCAlYl%XXA AUKALSPAAILALEKEYANK CCAMAAAGCATTGTCTCCTATC

GCAAATGGTAAACACG

CI’CCCOCTGDAAAAGGAGTACGCCAACAAAA

KLAAYVFPGRNLSLTVDADP AGC-ACGTC1CChGGCAGAAATTTA~mMCAGTAGACGCAGACCCAC

QAEISSKETLAENLTSLTLS A~~~~A~A~ARACAARCGCIW;CGGAGAAT NGARVILAKSAGEEQKLQII ATGGTGCCACGGnATK"IYjGC AAAATCCGCGGGTGAAGAG~AAAAG~TACAAATTATTG AVSNKGDLSFPAQQKSLIAL CCCrATCGAAT~~CAn*rAAG?TKTCTCCGCAGCAA

GRF 107 5220

Fig 1. Continued.

-ATTTTAGCAATATTCG

CTATTTTTATGTAATAATTTTATRARTGCGTTCAAAATAATAA'SCAAGTACTAATAGTGA

6120

TATTTTAAGGTCTGATTTTTACGTGATAATTCAGGAGACACAGAATGCGCATAAAAATAA

6180

CAGCATAAAACACCTTACCACCACCCAAGAATTTCATA ~TTTTTCAATGAAAA

6240

AA~TCGCGTRATATCTCACGATAAATA~CAl'TAGGATTTTGTTATTT~CACGA

6300

GTCCTTTGCACTTGClTACTTTATCGATAAATCCTACTTTT'N'AATGCGATCCAATCATT

TTAAWTTTW-

MDKKQVTDLRSELLD ATAAGAAGCAAGTAACGGATTTAAGGTCGGAACTACTCGATT

6360 ORF 109 6423

SRFGAKSISTIAESKRFPLH CACGTTTTGGTGCGAAGTCTATTTCCACTATCGCAGA4TCfbUACGTTTTCCGCTGCACC

6460

EMRDDVAFQI I N AAATGCGCGACGATGTCGCA'l'TCCAGACGAAACG

654G

D

E

L

Y

L

D

G

N

ARQNLATFCQTWDDENVHKL CTCGTCAGAACCTGGCCACTl-GCCAGACCTGGGACGAC~TGTCCACAAATTGA

6600

MDLSINKNWIDKEEYPQSAA TGGATTTATCCATTAACAAAi&CTGGATCGACAAAGAAGAATATCCGCAATCCGCAGCCA

6660

IDLRCVNMVADLWHAPAPKN TCGACCTGCGTTGCGTAAATATGGTTGCCGATCTGTGGCATGCGCCTGCGCCGAAAAATG

6720

GQAVGTNTIGSSEACMLGGM GTCAGGCCGTXGCACCAACACCAT"XGTTC~CCGAGGCCTGTATGCTCGGCGGGATGG

6'780

AMKWRWRKRHEAAGKPTDKP CGATGAAA'XGCGTTGGCGCAAGCGTATGG~GCTGCAGG~XAACCAACGCATAAACCAA

6840

NLVCGPVQICWHKFARYWDV ACCTGGTGTGCGGTCCGGTACAAATCTGCTGGXZATAAAT

6900

EL R E I PM R PGP L FM D P K RM I AGCTGCGTGAGATCCCTATGCCCCGGTCAGTTGTTTATGGACCCGAPACGCA'TGATTG

6960

EACDENTIGVVPTFGVTYTG AAGCCTGTGACGAAAACACCATCGGXGTGGTGCCGACT'M'UXCGTGACCTACAC'XGTA

7020

NYEFPQPLHDALDKFQADTG ACTATGAGTTCCCACAACCGTGCACGATGCGCTGGATlUATTCCAGGCCGATACCGGTA

?080

IDIDMH IDAASGGFLAPFVA TCGACATCGACATGCACATCGACGCn;CCAGCGGTGCCTTCACCGTTCGTCGCCC

7140

PDIVWDFRLPRVKS ISASGH CGGATATCGTCTGGGACTTCCGCCTGCCGCGTGTGAAATCGATCAGTGCTTCAGGCCATA

7230

KFGLAPLGCGWVIWRDEEAL AATTCGGTCTGGCTCCGCTTGCGGCTGGGTTATCTGGCGTGACGAAGAAGCGC-XC

7260

PQELVFNVDYLGGQIGwFAI CGCAGGAACTGGTGTTCAACGTTGACTACCTGGGTGGTCAAAT'IGGTACTTTTGCCATCA

7320

NFSRPAGQVIAQYY ACTTCTCCCGCCCGGCGGGTCAGGTAZ.T"TGCACAGTACTAT

7361

Fig 1. Continued.

To test whether proteins corresponding to different QRFs could be detected, maxi-cell experiments were performed with fragments from pBGT3 cloned in pTZl8 and pTZl9 [26]. With plasmid pBGT300, harbouring the 413%kb NirzdII1 fragment from pBGT3 (see table I) that contained 0RF102, ORF103 and a small fragment of QRF106, two polypeptides of 28 kDa and 85 kDa were expressed (fig 2, lane C). The 28 kDa polypeptide could be the gene product of ORF102, a peptide of 27.5 kDa as deduced from the nucleotide sequence and the 85 kDa polypeptide could be the product of 0RF103 which encodes a 86.7 kDa peptide. With plasmid pBGT303 harbouring the 2762 kb BgflI fragment from pBGT3 (see table I) which contains the entire ORF’36 and small fragments of ORFlrl)3 and 0RF107, three polypeptides of 82 kDa, 60 kDa and 56 kDa were expressed (fig 2, lane B). The 82 kDa polypeptide could be the gene product of 0RF106 which encodes fcr a 8 1 kDa polypeptide. The 60 kDa and the 56 kDa polypeptides are probably degradation products of the 82 kDa polypeptide

ig 2. Autoradiogram of (%) methionine-labelled proteins in E Eli SEXN?!? maxi-cells containing pTZ: 9 (lace A), plasnid pBGT.982 (lane B). or pBGT300 (lane C). 14C labelled proteins were used as molecular mass markers. Proteins were resolved in a 12.5% polyacrylamide-SDS gel and identified after fluorography.

828 Sequence comparisons

BLAST database searches with the gene products of the different ORFs were performed using the BLASTP [36] program. No similarities were found between the proteins encoded by 0RF102, 103, 106, 107 and 109 and the gene products of pqqi, -11, -111,-IV of A calcoaceticus [ 101. No significant homologies were observed either between the gene products of the ORFs and the proteins encoded by the pqq ABCDE genes from K pneumoniae [ 111. The 82 kDa polypeptide encoded by ORF 106 showed 17% identity with the gene product of pqqF from K pneumoniae [ 1 l] and several proteases like the human insulin-degrading enzyme (22% identical amino acids) [42], the mitochondrial processing protease enhancing protein precursor (PEP) from Neurospora crassa [43] and Saccharomyces cerevisiae [44] (20% and 21% identical amino acids respectively), and with the protease III from E coli (15% identical amino acids) [45]. All these identities were observed in the N-terminal part of the ORF 106 gene product (amino acids l-200) (fig

3). Similar identities with these prokaryotic and eucaryotic protease were previously reported for the gene product of pqqF of K pneumoniae [ 1 I]. Thus, the proteins encoded by E coli 0RF106 and KpneumoniaepqqFgenes could belong to a large family of zinc proteases. The gene product of 102 shows significant identities with transmembrane proteins involved either in transport or in secretion. No significant similarities were found between the gene products 0RF103 and 0RF107 and proteins in the databases. ORF109 was shown to contain 1005 bp of the gadI gene encoding for 329 amino acids of the E coli glutamate decarboxylase [4 11. Identification of ORFs involved in complementation pqqE and pqqF mutants from organophilum

of

To gain insight into the role of ORFs in complementations of pqqE and pqqF mutants of M organophilum, pBGT3 derivatives containing deletions or insertions were built and tested for complementation (fig 4). A non-polar insertion of

ORF,106 ide,huxnan ptr,ecoli kapqqfa mpp2Jeast mpp2_neucr

.................... MEIIMRNLCFLLTLVATLLLPGR. T;;T;&~LLHPALPSTFRSVLGARLPPPERLCGFQKKTYSKMNNPAIKRIG .... ..MPRSTWFKALLLLVALWAPLSQAETGWQ.............PI Q .................................................. .................................. ..MFSRTASKF~TR R ...................... JUURRLALNLAQGVKARAGGVINPFRR

ORF,106 ide,human ptr,ecoli kapwfa mpp2_yeast xnpp2,neucr

.LIAAALPQDEKL YPHAHPkDQVNLW NHITKSPEDKREY SDPTTDKSSAAL.. ETIRKSDKDNRQYQAIRLDNGMWLLVSDPQAVKSLSAL. ..MTLATRTVTLPGGLQATLVHQPQADRAAALA..RV~GSHHE LLST;SSQIP.GTRTSKLPNCZLTIATEYIPNTSSATVGIF..VDAGSRAE GLATPHSGTGIKTQTTTLKNGLTVASQYSPYAQTSTVGMWE

ORF,106 ide,human ptr,ecoli kapwfa xnpp2Jeast mpp2,neucr

EDNELwm FVgIIMMFNOTKTWPGNKVIETFESMGLRFGR PPNIAGLSEFCEXMLFLGTKKYPKEN...EYSQFLSEHAG PEAYQGLAEYL SFMGSKKYPQAD...SLAEYLKMHGGS PSRFPGLAELLEELLFYGGERYQDDD...RLMGWVQRQGGS NVKNNGTAEFLEaLAFKGTQNRSQQGIELEXENIGSHL.... TDETNGTAHFLEHLAFKGTTKRTQQQLELEIENMGAHL....

ORF,106 ide,human ptr,ecoli kapwfa mpp2yeast mpp2,neucr

nrrQvSLPTTQKQNLQQVMAIFSEWSNAATFEKLEVDA.ERGVITEEWRAH TNY.. .YFDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKN TAF.. .YLEVENDALPGAVDRLADAIAEPLLDKKYAERERNAVNAELTMA SAF.. .FFEVAADALADGVARLQEMLQAPLLLREDIQREVAVIDAEYRLI TVY.. .YAKSLQEDIPKAVDILSDILTKSVLDNSAIERERDVIIRESEEV TVY.. .FAKALNEDVPKCVDILQDILQNSKLEESAIERERDVIL~SEEV

TSYDE TSGEH TAPYR TLARH TSREN TSREN

Fig 3. Alignment of the N-terminal part of the gene product of ORF106

with the N-terminal part of human insulin degrading enzyme (idehuman) [42], protease III of E coii (ptr-ecoli) [45], pqqF gene product of Klebsiella pneumoniae (kapqqfa) [ 111, mitochondrial processing enhancing protein from Saccharomyces cerevisae (mppZyeast) [44], and mitochondrial processmg enhancing protein from Neurospora crassa

(mpp2-neucr) [43]. Bold letters indicate residues that are identical to ORF106 gene product in all the homologues aligned.

analysis of pBGT3 derivatives. All these plasmids were transferred from E coli S 17.1 strain to M organomatings as described in Materials and methods. After conjugation, mating mixtures were plated on Mac Lennan minimal medium with methanol as carbon source. Abbreviations used in the table: C, complementation; -, no complementation; Bg, &/II; E, E~aRI; W, HindHI; iP,PstI. Symbols: open triangle, km cartridge; solid triangle, Tn5. . Map and complementation

phifum mutants using biparental

site positioned in a kanamicyn cartri T40) did not affect the complemenQRF 183 (plasmid p tation abilities of pB 3. According to this result, the gene ’ product of OFR103 is not involved in the complem of ~4qE and ~4qF mutants of M organophilrrm. In a partial deletion of ORF107 and the entire deletion of ORF109 abolished complementations of both pqqE and pqqF mutants of M organophiium. As reported above, ORF109 is only partially contained in pBGT3 and therefore presu ly can not play a role in complementation. This latter point was confirmed by the normal complementation pattern obtained with pBGT322? a plasmid which contains a Tn5 insertion located in 0RF109. A Tn5 insertion located in ORF 107 (plasmid pBGT369) abolished complementation of both pqqE and pqqF mutants of M organophiium, suggesting the requirement of 0RF107 for complementation of both classes of mutants. The deletion of the 150 bp Hind111 fragment, contained in ORF 106 (plasmid pBGT 10) or its replacement by a kanamicyn cartridge (plasmid pBGT30) abolished also complementation of both pqqE and pqqF mutants of A4organophilum. Therefore, ORF 106 as 0RF107, is involved in complementation of pqqE and pqqF mutants. On the other hand, 0RF106 taken separately could not complement the mutants since the &III fragment of pBGT3, containing the entire QRFl06, cloned in pLA2917 (plasmid pBGT9) did not complement either

pqqE or pqqF mutants. According to these results, the complementatiorn of pqqE and pqqF mutants requires the activ-

ity of both ORF 106 and e products. Other ties offered by the deletions built according to restriction map did not provide the possibility to determine whether 0RF102 was involved in complementations of pqqE and pqqF mutants of M organophilum. Phenotype of mutants containing chr-omosomal insertion RFs contabled iu pBCT3

In order to investigate the involvement of the different ORFs in PQQ biosynthesis and/or in PQQ processing in E coli, DNA fragments with insertions in ORFs 103, and 107 (fig 5) were recombined with the chromosome of E ct/li V355 ed in Materials and methods and then trznsferred as d producing strains (EF strains) by Pl transductions. into The correct localizations of the insertions on the chromosome were visualised by hybridisation experiments of appropriate probes with chromosomal DNA from strain EF260 or from its derivatives containing chromosomal insertions in ORF 103 or ORF107 (data not shown). The EF derived strains harbouring chromosomal insertions in ORFl03 and 0RF107 were first tested for growth on lVlacLennan minimal medium with glucose or giuconate as carbon sources. Strain EF260, used as positive control

830

Fig 5. Map of insertions of Cm resistance (open triangle) and Ap resistance (solid triangle) containing fragments in 0RF103, and ORF107. Abbreviations: Bg, BgAI; E, EcoRI; H, HindIII; P, PstI.

utilisation, oxidises glucose into gluconate by the PQQ-dependent holo-glucose dehydrogenase (holoGDH). Strains FB8A@ galP was used as negative control for growth on glucose, and as positive control for growth on glucose with added PQQ. As shown in table II, chromosomal insertions in ORF 103 and 107 increased the doubling time about 2.6 times in glucose minimal medium as compared with the parental strain EF260 grown in the same conditions. These insertions did not affect growth in gluconate minimal medium and therefnrp eaaVnh-e-v,,-~ vub I cu -1 ,3 owing down effect is specific for the rV*” thm glucose oxidation by the PQQ-dependent GDH. In order to investigate if strains harbouring chromosomal insertions were impaired in the assembly of PQQ with the glucose dehydrogenase, the strain FBS &ts gal P and its derivatives containing either an insertion in 0RF103 or in ORF 107 were tested for growth on glucose minimal medium supplemented with PQQ. In table II, the results show that the chromosomal insertions in ORF103 or in 0RF107 did not affect the growth of a Apts gal P strain on glucose minimal medium supplemented with 1 nM of PQQ. Surprisingly, the doubling time on glucose minimal medium in the presence of PQQ is increased about 1.3 times for EF260 derivatives containing chromosomal insertion in 0RF103 or in ORF107 as compared with EF260. As reported above, the

for glucose

Table II. Doubling times (in min) of strains, containing chromosomal insertions in ORFs 103 or 107 grown in minimal medium with glucose or gluccnate as carbon source. When required, PQQ was added at the final concentration of 1 nM. EF 260 strain is a Pqq+ derivative mutant of FB8 Apts gaZP. The experiment was repeated several times. A representative result is shown. Gluconate

FB8 Apts gulp FBS Apts galP. 103 FB8 Apts gafP. 107 EF 260 EF 260.103 EF 260.107

tS 85 85 85 85

Glucose

Glucose +

No growth

100

No growth No growth 110 290 290

100 100 110 140 140

PQQ

gene product of 0RF103 was not involved in complementation of /qqE and ~qqF mutants of ha organophiltim. Consequently, the phenotype generated by insertion of an ampicillin cartridge in ORF103 can be explained by a polar effect on expression of ORF 106 and 0 F 10’7located downstream in the operon. According to th henotype generated by its disruption, the gene product of 0RF107 could be involved in a late step of PQQ biosynthesis rather than in its transfer from the cytoplasm or its association with the periplasmic apoglucose dehydrogenase (apoGDH). Indeed, a late precursor of PQQ biosynthesis could bind the apoGDH and act as a less effective prosthetic group than PQQ. Moreover, such late precursor could also decrease the binding of exogenous PQQ with the apoGDH no conclusion can be drawn with regard to thesis since disruption of this ORF is apparently toxic for the bacteria. The function of ORF106 and ORF 107 in E coli is still unknown since they can not be considered as PQQ biosynthesis genes like the ~49 genes isolated from A calcoaceticus [ lo], K pneumoniae [ 111, M organophilum [ 5 1, or M extorquens [6]. The gene product of ORF106 and ORF 107 could be involved in another process than PQQ biosynthesis in E coli and their activity could mimic the catalytic activity of the pqqE and pqqF genes of Af organophilurn. Such an activity has also been recently hypolhesised in E coli to substitute for PqqF of K pneumoniae 1141.

Acknowledgments This work was supported by grants from the Centre National de la Recherche Scientifique (URA 1129) to A Danchin.

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