Molecular cloning and characterization of the pyrB gene of Lactobacillus leichmannii encoding aspartate transcarbamylase

Bi,,;,,'k,imic (1996) 78, 3-13 C) Soci6t6 fran~;aise de biochimie el biologic mol6culaire / EUsevier, Paris

Molleculav cloning and characterization of the pyrB gene of Lacgobaciglus geichmannii encoding aspartate transcarbamylase J Becker, M Brendel* Institut fl~r Mikvobiolo~ie. JW Goethe--L;;~i,cr.,:i'=~t. Theodo;" Sie;n-Kai 7. Haus 75, 60596 FrankJi~rt/Main. Germany ¢Received 21 February 1995; accepted 3 August 1995}

Summary --- The Lactobaciilus leichmamtii pyrB gene, encoding pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase), was cloned from a partial genomic library lying on a 1468 bp SalI/Bs1XI fragment. The predicted polypeptide sequence extending over 351 amino acid residues (M~ 39 855 Da) was compared to those of various other orgamsms revealing clear identities towards them and important conservative stretches, implying lhat these proteins are closely related. Transcriptional initiation wa.., mapped by primer extension and occurred 54 bp upstream of the pyrB open reading frame (ORFL Northern blot analysis indicates that the pyrB gene is transcribed as a single mRNA and not together with the following overlapping pyrC gene as a bicistronic mRNA. At high copy number the pyrB gene of L leichmannii seems to be lethal for its E coil host; inserted in a low copy vector it complements the uracil auxotrophy of an E coli pyrB mmant which shows distinct ATCase activity in the cell extract. With an excess of uracil in the growth medium the gene is apparently repressed and no ATCase activity can be measured. aspartat~, ~ranscarbamy|ase / pyrB / pyrimidhle biosymhesis / Lactobacillus leichmannii / DNA sequence

Introduction The de novo synthesis of UMP, the precursor of all pyrimidine nucleotides, is catalyzed by six enzymes encoded by the pyr genes ( A - F ) which are common to almost all organisms. However, the genetic arrangement of the coding DNA sequences conlrolling tl3ese enzymatic reactions varies greatly and lhe number of the encoding genes decreases from prokaryates to higher eukaryotes. In most prokaryotes like E ('oli or S typhimurium the individual pyr genes are either scattered over the genome [l, 21 or arranged in operons (eg in B subtilis or B caMolyticus, [3, 4]) whereas in higher eukaryotes gene clusters have evolved from fusions of formerly independent genes so that the first two or three con'esponding enzymatic activities ate located on a multifunctional protein [5] whereas the last two steps are associated within a bifunctional prolein [6]. In L leichmannii, however, the first enzyme of this pathway, the carbamyl phosphate synthetase, encoded by the pyrA gene is thought to be lacking [7] so that aspartate transcarbamylase (ATCase, carbamoyl phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2.) encoded by the pyrB gene is supposed to

*Correspondence and reprints

catalyze here the first reaction committed to pyrimidine nucleotide biosynthesis, the carbamylation of the amino group of aspartate by carbamyl phosphate. This enzyme has been extensively examined in several or~,amsms, and in E ~'oli and S t~7~himurium it consists of catalytic and regulatory subunits possessing allosteric control sites [8, 9]. In S cerevisiae ATCase is covalem[y linked with carbamyl phosphale synthetase [10[ and in mammalians it is one of the enzymmic domains of the trifunctional CAD protein i l l [ . The con'esponding gene, which in some cases is part of an operon or gene complex, has been thoroughly studied, especially in E coli where it is regulated by an UTPmodulated attenuation control mechanism [12]. Excepting a study concerning the dependence of ATCase activity on the presence of uracil during growth [7], nothing is known aboul the ATCase of L leichmannii and its encoding gene. Therefore we cloned the L leichma;mii p.v~B gene in order to obtain first information about its DNA sequence and the encoded gene product.

Materia|s and methods Strains The E coli KI2 strains used for transformalion and amplification of plasmid DNA were JMi09 (recA|, endAl, gy;'A1, rhi, hsdRl7, supE44, relA1, A(/ac-proABL iF', traD36, proA+B+,

laclqZAM 151), DH5tx (80d lacZ AM i 5, emtA I, recA I, hsdR 17, (rk-, m~+), supE44, thi-l, ~,-, gyrAI, relAl, A(lacZYA-argF), U 169), Hfr3000 YA289 (thi- 1, pyrB289, relA I, spoTl), AT2535 (ureA2 or toni4, lacYi, tsx-23 or tsx-25, supE44, ~,-, hisGl, r]bDl, purFI, lpsL8, 9 or 14, malT! (kr), .~y/-7, mtl-2, argHl, thi-l, pyrB59), MAI008 (thi-I, pyrC46, relAl, iacZ43, ~,-, spoTl) and Si31256 (araD139, A(lac)U169, rpsL, thi-, ApyrB). The last four strains were gifts from Barbara M Bachmann (E coli Genetic Stock Center, Yale University, New Haven, USA) and Kaj Frank Jensen (Institute of Biological Chemistry B, University of Copenhagen, Denmark). B subtilis strains IA392 (ILvrB, trp2) and 1A394 (pyrC, trp2) were kindly provided by the Bacillus Genetic Stock Center (Ohio State University, Columbus, USA). E coli strains were grown on Luria broth (LB), SOC medium [131 or minimal medium M9 described by Glansdorff [141 at 37°C. B subtilis was grown or, LB or supplemented Spizizen's minimal medium !151. Ampicillin (final concentration: 40 l.tg/ml), tetracycline (at 12.5 tug/ ml), kanamycin (at 50 tug/mi), chloramphenicol (at 5 ~g/ml), and other supplements (eg thiamine, arginine, histidine, adenine, tryptophane, and uracil at final concentrations of 20 tug/ ml resp 50 tug/ml for B subtilis) were added when required. L leichmannii DSM20076 was used for the isolation of chromosomal DNA, RNA, and determination of enzymatic activity.

Preparation and analysis t~'DNA The CTAB method [22] was used |br the preparation of plasmid DNA from E coli. B subtilis plasmid DNA was isolated as described by Roddguez and Tail 123]. Restricf~m, dephosphorylation of 5' ends, and ligation of DNA were carried out as recommended by the manufacturer (Boehrmger, Mannhelm, Germany). Agarose gel electrophoresis was performed as described by Sambrook et ai [241 and DNA fragments were eluted from agarose gels by the fast flush method [25] or using a Biotrap electroelution apparatus (Schleicher & Schull, Dassel, Germany). DNA was radioactively labeled using the "random primed DNA labeling kit' (Boehringer, Mannheim, Germany). A vacuum blotter (Pharmacia LKB, Freiburg, Germany) was applied for blotting DNA on nitrocellulose filters and the DNA-DNA hybridization was achieved according to Southern [261 and Maniatis et al [27].

Transfi~rmations E coil was transformed according to the slightl) modified electroporation procedure described by Ausubel et al [13] and B subtilis by following the protocol of Klein et al [28] except that HS- and LS-medium were supplemented with 50 tug/m! resp 5 tug/mi uracil.

Plasmids

Construction of a partial j,'cnomic library of Lactobacillus leichmannii

pBluescript SK ÷ (Stratagene, Calitbrnia, USA) was used for the construction of the partial genomic library in E coil DH50~. Plasmids pJB9, pJB904 and pJB903 isolated from a Sau3A genomic library [16] are also derivatives of pBluescript with approximately 2.75, 2.5 resp 6.5 kb inserts which comprise the entire pyrC gene and a part of the pyrB gene. Vector pBR322 [17] was used for the amplification of the complete L ieichmamaii pyrB gene in E coil and the low-copy-vector pCKIg, a derivative of pUC 19 with a pSCIOI origin which confers resistance to kanamycin (KeU and Spratt, unpublished results), for the expression o1' this gene in E coll. For the transfl)rmation t,f B suhtilis the E coli-B suhtilis-shuttle vector pHVi432 il81 was tlsed.

High molecular chromosomal DNA from L leichmannii was double-restricted with BstXl and Pstl resp BstXl and Sail and separated by agarose gel electrophoresis. Fragments of suitable size, deduced from Southern blot analysis with radiolabeled probes originated from the already known part of the pyrB gene 1161, were eluted from the gel and cl,3ned into equivalent restricted pBluescript. E coil DH5ot was transtbrmed with the ligated DNA and plated on LB plates supplemented with ampicillin and X-gal (5-bromo-4-chioro-3-indolyl-~-D-galactoside, 80 tug/roll for blue/white screening. About 6000 white colonies were picked on fresh LB plates and incubated overnight at 37°C. For longer s~otage the colonies were pooled in liquid LB with I(1% glycerol and frozen at-70°C.

Isolation of high molecular gemmtic DNA

Chining and sequencing of the pyrB gene

Genomic DNA of L h,ichmannii was purified according to Williams et al 1191 with the following main modifications: cells in the mid- to late logarithmic phase were washed twice in icecold 0.7% NaC! solution and, after freezing and thawing in water, the cell suspension was heated for 10 min to 70°C and cooled to room temperature to inactivate possible nucleuses. Protoplasts were formed by incubating the cells in a 3% 4amino salicylate suspension with !.2 mg/ml lysozyme. Cells were lysed by the addition of SDS to give a final concentration of i% and proteins were digested corepletely by adding 0.55 mg/ml proteinase K instead of pronase. For rapid isolation of chromosomal DNA the slightly modified technique of Mannur 1201 was used.

The white colonies of the partial genomic library were directly transferred on HATF (Millipore, Eschborn, Germany) or nitrocellulose (Schleicher and Schueil, Dassei, Germany) fiiter~ and incubated on fresh LB plates for at least 3 h. For cell lysis, denaturation, and neutralization the filters were treated as described by Sambrook et al [241, dried and baked at 80°C for 2 h. Prior to hybridization with a DNA probe, the filters were rinsed in prewashing solution (50 mM Tris hydrochloride (pH 8.0), I M NaCI, I mM EDTA, 0.1% SDS) at 42°C for 90 min ar,d cell debris resp protein were rubbed off thoroughly. Colohy hybridization followed the protocols of Maniatis et al [27] and Southern [26]. The DNA probes consisted of two [tx32P]dATP labeled EcoRl fragments with lengths of 146 resp 144 bp that had been cut out from the incomplete pyrB ORF of pJB904. DNA was sequenced according to Sanger et ai [29]. Plasraids pJB9, pJB904, pJB6, pJB8, pJB7, and subciones of the last one were the source material. The universal and T3 primer were used to initiate the sequencing process. In orJer to read the sequence entirely in both orientations, oligonucleatides had to be synthesized to serve as primers which were all designed using the data from each successive round of sequencing. T7

RNA isolation Total L leichmannii RNA was isolated by the hot phenol protocol described by Shaw and Clewell [211 and E coil RNA by the rapid isolation protocol of Ausubel et al ]131. The quality and concentration of the isolated RNA was estimated by denaturing and non-denaturing agarose gel electrophoresis.

DNA polymerase sequencing kit was from Pharmacia/LKB (Fretburg, Germany) terminator cycle sequencing kit from Appfied Biosystems (Foster City, California, USA) and [~35S ]dATP resp ~o03PIdATP from Amersham (Braunschweig, Germany) DNA and protein sequences were compiled and analyzed with DNAS|S/PROSIS computer programs from Pharmacia/LKB (Freiburg, Germany). Homology searches were performed with Swiss-Prot R23.0 (European Molecular Biology Laboratory) kindly provided by KD Entian.

amount of enzyme fl~al catalyzes lhe formation of n pmoB of carbamyl aspanale per minute under these reaction conditions. Specific enzyme activity is given m units of activily per mg of protein. Protei~t concentn'ations were assayed by the method of BradR-~rd [331 with bovine serum albumin as the s:andard.

Polymerase c~min reaction

Cloning and sequencing ¢~'the pyrB gene

PCR techniques were carried out as described by Ausubel et al [13] with slight modifications. For each reaction 2 btg L ieichmannii chromosomal DNA was mixed with 300 ng of sense (5'-CAAACATTCGAATTGACAGGA-3') and anti-sense primers (5'-CGCCTrCCTGGTAAACCAGGCCG-3) respectively, dNTPs (2 raM), MgCl: (4 raM), 10 ~l amplification buffer (10 x) and 1 ~tl Taq DNA polymerase (4 units/iul, Eurogentec, Seraing, Belgium) in a final volume of 100 ~tl.

Attempts to clone the entire pyrB gene via cornplementation of an E coli-pyrB-auxotrophy by transforming the corresponding mutant strains (pyrB289 or ApyrB) with plasmid DNA isolated from a l. leichmannii Sau3A genomic library that ,~as established in the vector pBluescript [16] and plating the transformed cells on minimal m e d i u m without uracil were unsuccessful. Theretbre we screened this gene bank by colony hybridization with a radiolabeled probe originating from the already known (C'-terminal) part of the pyrB open reading frame found by sequencing the approximately 2.75 kb insert of pJB9 which bears the entire pyrC gene [16]. By screening approximately 40000 colonies we were able to isolate some clones which extendea the pyrB O R F for 177 bp; they cou!d also be found via complementation of E coil pyrC mutants [16]. For cloning the entire pyrB gene we hence decided to construct two partial genomic libraries of L leichmannii in which the entire gene should be amplilied. By screenir, g 6000 different clones of these parliai libraries via colony hybridization we detected cnly one positive clone carrying a plasmid (pJB6) with a Pstl/BstXl-insert of approximately 3 kb which we suspected to contain the complete pyrB ORF. Subcloning of pJB6 by Sail digestion and religation of an approximately 4.4 kb Sa/|-fragment comprising pBluescript and a 1.47 kb sabfragment of the pJB6 insert yie!ded plasmid pJB7. However, neither of these plasmids conferred to transformed E coil pyrB mutants the ability to grow on M9 minimal m e d i u m without uracil nor could we detect ATCase enzymatic activity in the respective cell extracts. Sequencing of both DNA strands of pJB7 revealed 96 bp of the N'-terminal part of the pyrC gene [16] on the 3' end of the 1468 bp insert. Two additional ORFs of 402 resp 582 bp length were also found on distinct frames and both of them showed strong identities towards D N A sequences of known pyrB genes of other organisms. Therefore we assumed that these two ORFs belonged together and were probably separated by a frame shift mutation upstream of the 582 bp O R E By e m p l o y i n g polymerase chain reaction with c h r o m o s o m a l L leichmannii D N A as template and HindIII/Apal digestion we generated a 536 bp fragment which covered the critical area of the putative frame shift; it was iigated in HindlII/Apa~-cut

Analysis of mRNA The blomng of RNA on nitrocellulose fil~ers was carried out by vacuum blotting (Pharmacia LKB, Freiburg, Germany) and DNA-RNA hybridization was achieved according to Ausubel et al [13] and Maniatis et al [27] with slight modifications concerning the hybridization buffer (50% formamide, 5 × SSC, 50 mM sodium phosphate (pH 6.5), 200 pig/rid salmon sperm DNA, I × Denhardt's solution) and using the 536 bp Ncol/ ApaI-fi'agment cut out from pJB8 as probe. The 5' termini of pyrB transcripts were mapped by primer extension as described by McNeil and Smith [30] and Schenk-Gr5ninger et al [16] with some modifications: 50 ng of an appropriate 21 mer oligonucleotide primer (5'-TTTCGACAACGACTTGGTTTT-Y) and cellular RNA (80 pg) isolated from L lei~'hmamffi or lhe recombinant E coil YA')Ro strains were used. For hybridization the sample was at first heated at 90°C for 2 rain, then at 65°C for 10 rain and finally incubated at 42°C for 60 ram. For the prhner elongation reaction Ict3:PldATP instead of i eDsS]dCTP and 40 U AMV reverse transcriptase (Mobit~y:, G6ttingen, Germany) was used. The reaction mixture was incubated at 42"C for 60 min and then stopped by adding 0.5 ~i 0.5 M EDTA. After incubation with ! gtg RNase A at 37°C for 60 min the primer extension products were precipitated with ethanol, desalted and dried. The st*topic was analyzed ~tl'ter denaturing at 80°C for 2 min by running on a 6% denaturing polyacryiamide sequencing gel alongside an appropriate DNA sequen,cing ladder that was generated by using the same primer as in the primer extension reactions.

Preparation of cell extract and enzyme assay L leichmannii was grown in 250 ml MRS medium at 37°C for more than 20 h. E coil was grown in 100 mi LB medium at 37°C overnight. Cells were harvested by centrifugation, washed twice in an equal volume of 50 mM Tris (pH 8.1), resuspended in 5 ml of the same buffer and frozen at -70°C in Eaton presses [31]. Crude extracts were prepared by disrupting cells at a pressure of 200--250 bar and centrifuging the thawed extract at 12300 g and 4°C for 15 rain. The supernatant was stored at -70°C with 10 ~M PMSF added. The ATCase assay was carried out at 30°C for 1 h and contained 100 mM Tris (pH 8.1 ), 40 mM L-aspartate, 10 mM carbamyl phosphate, and enzyme to yield a final volume of 1 mi following the colotimetric assay [32]. One unit of enzyme activity is defined as the

ResuRs

pBluescript yielding pJB8. Sequencing the DNA upstream the 582 bp ORF presented an additional base pair (G/C at position 705) that fused both ORFs into one. The resulting complete ORF is arranged in reverted orientation towards the lac promoter of pBluescript and consists of 1053 bp. Restriction mapping confirmed the restriction sites of pJB7 presented in figure 1. Suspecting that overproduction of L leichmannii ATCase, causing accumulation of carbamyl aspartate, might be toxic for the E coli host, we combined the pyrB and the pyrC gene by ligating the SalI/Apal fragment of pJB7 and the Apal/Clal fragment of pJB9 into vector pBR322 hereby generating plasmid pJB 11 (fig 3). This plasmid, at 15-20 copies/cell, should have a much lower copy number than pBluescript. The frame-shift mutation in the pyrB ORF was removed by replacing the NcoI/ApaI fragment of pJBll with the corresponding fragment of pJB8 (---> pJB1 la; fig 3). E colipyrB auxotrophic ceils bearing this plasmid were able to grow on LB medium and minimal medium with uracil but not on minimal medium without uracil. In cell extracts made from YA289(pJB1 l a) grown on LB medium or minimal medium supplemented with uracil no ATCase activity could be measured. Therefore the inserts were cut out from pJBl la by Sall/PstI-digestion and ligated into the low-copy vector pCKI9 (--5 copies/cell) gene-

5'

1 TCGACTCCT'rAGCTCGATTATACCGCAATCACTTCAGTA.AGCOGTTGAAA..tCGGTTTT..~CTT~TT~TAC ~adZZZ 72 `~AAA`~TTCCCTG~T1`T~CGATAA`~A~GCTT~A~TTAA~AGGGC~AAGTCT3~TATAG~T~AG`L~A`~TA 143 TATGAATGACC'Im~A~ulI"~rAG~CCCGTGAGGCTA~IGA.IGGCAGCTCA~A1"CCAT~TATAAACA~G~T -36 ~ndlll-~O "1 214 TCTTGGC'~-AACATTCGAA~4C~-~tAAGACCGG6GAGGAAGCTT~GG~CAGTTT.~GCAGACTTTGAG 2 8 5 AGACCA~AGACAC~.~'~a~CTGAGTGTATAAA

&TG GTC ~PG Ct~ AAG ~ A 1"tT TCG 1 H e r V a l Phe L~u Leu Lya Lou Phe Set

3 4 6 AAA GGA I'GC CAA ~TC ATC ATG &CC ACT GTT AGC CA.~ ,~C C,~ GTC GTT GTC GAA 10 Lys G I y Cy~ Gin ~le l i e ~e~ Thr Thr V a l S e t Gin X s n Gin Val V&l Va/ GIu

400 ACT G.~L~ GAC ~ G C,~. QAC AAC 1~G CTC CGT CTG CCT ~.aC TTT GTC AGC GTT GAA 28 T h r GIu A ~ p L y a Gin A s p A~n Leu Leu Arg Leu Pro Tyr Phe Val S e r V&l G I ~ 4 5 4 CAA CTG AG~ GCC GAC GAC GTG CTG CAC CTG 46 O~n Leu S e t AI& A~p Aop V~l Leu Hi8 Lou Nael 508 AAC GGC ~ GAA GTA CCA GCC I~I'G AGC CGG 64 ASh G l y G I ~ O l u V a l P r o AI& Leu S e t Ar~

CTG C ~ ~ GCC C ~ TAC 1~'C A~G Leu Gin .-k~ AI& Gin TFr Phe Lys

CCA ATC ~'~C TGC ACC AAC ATG T~C Pro l i e L>bo CyS T h r A~n } l e t Phe

562 T t T G,~ ~U~C TCA &CC CGG &CC CAC ACC AGC GAA ~ GCT GAA AGA AGG CTG 62 Phe G I u AS~ S e t Thr Ar~ Thr HLs Thr S e t Pbe GIu V&I AI& 61u Ar~ Art; Leu 6 1 6 GGC TTG ACT GTA &TC CCA T't'~ GAC CCA 100 GIy Leu T b r v a l l i e Pro Phe &ap Pro NcoI 6 7 0 AAC CTC ?AC GAC ACT QAA CTG ACC ATQ 118 A~n L e u ~ A~p T h r G I u L e u T h P ~ e ~

TCC CAC TO? TCA GTC A.IC .~G GGG G~; S e t E t a See Set V a l Ash Lys G l y GZu GCA TC~ CTQ GGC A'~I' G~u~ CTC AGC GV~ AI& S e t L ~ GIy Zle GIu ~eu S e t va~

72~ ATC AGA CAC CCG GAA AAC GCC YAC TAC AAC ~AA ATC ATC CGC CCA AAG GAA GGC 136 ~ l e A r ~ B~a Pro GZu Aa~ A l a T y r ~ y ~ Ash Glu I1~ l i e ~r~ Pro Lga Glu Gty

7T6 CAG CAC ~ C ~ &TG GGC CTG GTT AAC GCT GGT GAC G G T T C C 154 Gin ELa Leu Gin He~ Gly L e u Vml Ash A l a G l y ASp GIy S e t £coal 832 TCC C~, AG~ ATG CTG GAC ATG ATG ACC ATC TAC AAC GAA ~l'C 172 Set GZn S~r He~ Leu Asp Ne~ ~e¢ ThP l i e T y r A a ~ ~ l u Phe Seal 886 GGC 1"I'G ~ G ATC 4TG ATC GTC GGT GAC TTG ACC A,IC tCC CGG 190 G l y Leu L y ~ l i e ~ e t I l e v a l GIy Asp Leu ? h r .tan Set t r ~ 946 AAC ATG GAA ATC TTG AAT ACC CTG 2 0 8 Amn N e t Gl~ I l e Lau k~n Thr Leu £co~1 ~94 TAC ~ TAC AAC GCT GAG GAA l~rc 2 2 6 T y r T r p T y ~ A S h & l ~ Glu Glu PhO

GGC CAG CAC CCA GI~ Gin HZs Pro GGC CAC ~rc GAC GIF ~£s Phe Asp GTT 6CC CGG TCA Val Ala Arg Set

GGC GCG GAA G~C ~AC T T c TCA GOT CCG G~t Gl? k l & GIu V~l T y r Phe Set GIF 9 r o Glu AGC AAG TAC GGC ACT TAT G~C ~lG AAC A ? T S e t L y s T y r G I y T b r T y r V s l L y s Ash l i e

tO4e (;AC G a t ~ ATC CCA G ~ CTG GAC GTC 1~G &TG CTG CTC AGA GTT CAG CAC G ~ 2 4 4 ASp A s p GIu l i e P r s Olu Leu Asp V a l Leu ~ e ¢ Le~ Leu Ar~ Val ~In ~ts ~lu 1102 CGC CAC AAC GGG GCA OAA GCC ~ G ACT G ~ CAG CTC t'l"t QAT GCC .~,G GAC T~C 262 A~g H i s Asn O l y k l s Ol~ AIs L y s T h r Glu Gin L e ~ Phe Asp ;L& L y g A s p T y r 1 1 5 6 ~*~C GCT GCC TAC GGC C1~ 260 Amn A l a AI& T y r G I y Leu $~alApa~ 1210 ATC ATC ATG CAC CCG GG~ 2 9 6 l i e ~ l e ~ e ~ E t a Pro 0 1 ~

AAC C ~ CGC CGC TAC GAC A?G CTT AAG GAT GAC ~C: ASh Gin Ar~ Ar~ ~ y ¢ A s ~ ~e¢ Leu LFs ~sp Asp ~L~ CCA &TC ,~C CGC GGG GT~ G,~ TGG GAC GGT GAC CTG Pro ~ l e Ash At8 0 1 y vaL Olu t r p Aep GIy Asp Leu

1 2 6 4 QTT GAA GCA CCT AAG TCC CGC TAC GCT GTC CAA k ~ CAC ~lC GGG GTC TTT ~TC 316 V~l QIu A l ~ Pro Lya Set &r~ T y r A l a V&l Gin ~ e t 6 i s ~sn Gt¥ Val Phe Y&l

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1 3 1 6 &OA ATG GCC ATG ATI[' O ~ GCT GTT CTQ CGC r~A CG~ ~AG TTO GGA GGC CTT G&~ 334 AT6 ~ e ¢ &Ln ~ t t i e GIu A l s Val L~u AT8 O l y A r l Lys Leu G l y GIy Leu Gt~ sol g¢oKl ace 1 3 1 2 ~,q~ TGG CCA TTT TGC YAA AAA ACG G~_~GG T~T ~ , . . A ~ , ~ O OCG AAT TCA rCA

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L u , P",,, ~',,, ~, ,u_J~,, P"T,, P+"P, ,P~ Fig 1. Restriction sites and sequencing strategy of the 1.468 kb Sall/BstXI fragment of clone pJB7. The large open arrows represent the pyrB resp pyrC coding sequences. Arrows indicate the direction and extent of each sequence determination. Sequencing reactions utilized clones pJB8, pJB9, pJB904, pJB7, and restriction subclones with vector primers (solid arrows), as well as full-length clone templates with synthetic oligonucleotide primers (broken arrows),

Fig 2. Nucleotide sequence of the 1468 bp insert of pJB7 (corrected for an additional nucleotide at position 705) containing the L leichmannii pyrB gene and flanking regions. The sequence of the non-coding strand is numbered from the 5' end. The deduced amino acid sequences of the aspartate transcarbamylase and the partially available dihydroorotase are shown. The -10 and -35 hexamers and the Shine-Dalgamo sequence (RBS) are written in italic letters resp underlined and labeled. The transcriptional initiation site is marked by +1 and dyad symmetries are indicated by arrows. The ,~enbank (EMBL) accession number for the nucleotide sequence reported in this paper is X84262. rating p J B l l l which complemented the uracil auxotrophy of E coil pyrB mutants in minimal medium. E coli YA289(pJB 111) showed no enzymatic activity when grown on LB medium or M9 medium with uracil but ~vhen grown on M9 minimal medium without uracil for 3 - 4 days distinct ATCase activity

.

-

h .

--AE

Table L Identities of L leichmamlii asparlate ~ranscarbamylase wilh ATCases and OTCases oI" varioiJs other organi.~m~.

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identity (similaritvj Over a range with L leichmanni/ of amino ATCase acids of:

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PJ8 lla 7t.27 b p

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iw H $

Fig 3. Construction of plasmid pJBl l a. The closed bars represent L ieichmam#i insert DNA, the open bars vector DNA. The respective pyr gene sequences and the antibiotic resistance markers are indicated by arrows. Abbreviations of restriction enzymes: A, Apal; B, BstXl; Bin, BamHl; C, Clal; E, EcoRl; Ev, EcoRV; H, Hindlll; K, Kpnl; N, Nael: Nc, Ncol; Nr, Nrul; P, Pstl; Pv, PvuI; Pu, PvuII; S, Sail; Sa, Sacl; Srrt, Sinai.

could be measured (table |I). On the other hand replacing the defect Ncoi/ApaI fragment of plasmid pJB7 with the correct one and transforming E coli with this ligation mixtur,~ was not possible.

L ieichmamui ATCase B subtilis ATCase Hamster CAD protein Human CAD protein E coli ATCase S ~.phimurium ATCase S cerevisiae URA2 protein D discoideum pyrl protein S marcescens ATCase D melanogaster CAD protein P aeruginosa OTCase B subtilis OTCase E coli OTCase

100 (100) 47.4 (80.7) 30.6 (69.1) 30.3 (69.4) 29.3 (67.8) 28.7 (67.8) 26.9 (65.6) 26.6 (67.2) 32.2 (70.8)

351 306 314 314 311 314 323 338 236

24.1 23.3 22.5 24.1

320 309 307 199

(62.2) (63.4) (67.4) (66.8)

Inserting the SalI/BamHI-fragment of pJB 113 (figure not presented here), a derivative of pCK19 comprising just as well the entire pyrB and pyrC genes as pJB 111, into the SaII/BamHI-cut B subtilis-E coli shuttle vector pHVI432 yielded plasmid pJB213. B subtilis pyrB resp pyrC auxotrophic strains transformed with plasmid pJB213 could not be complemented to urac'lindependent growth on Spizizen's minimal medium without uracil in spite of the highly significant sequence homologies between the corresponding genes of the two Gram-positive organisms. Sequence analysis The 1053 bp ORF of pyrB encodes a polypeptide of 351 amino acid residues with a predicted molecular mass of 39 855 Da (fig 2) which is supported by results of protein purification showing a M, of about 40 000 Da for the purified ATCase of L leichmannii (unpublished results). The TAA stop codon of pyrB overlaps with the ATG start codon of the following pyrC gene. Analysis of the amino acid sequence deduced from the pyrB ORF showed considerable

Table If. Specific ATCase activities (units/mg protein) of cell extracts of L leichmannii and E coli transformed with plasmids pCK 19 and pJBl I 1. L leichmannii DSM20076 E coil YA289(pCK 19) E coli YA289(pJB 111 )

2.6 M9 medium + uracil

M9 medium - uracil

LB medium

0 0

O 0.81

0 0

identities towards the pyrB genes and lower identities towards the arg! genes (coding for ornithine carbamoyltransferase (OTCase) [34]) of diverse organisms (table I). Additionally highly conserved sequence motifs could be found, most probably belonging to the active site of the enzyme as judged by detailed examination of the E coli ATCase ([35-38]; fig 4). As deduced from analysis of the E coli enzyme, other conserved amino acid residues which do not directly belong to the catalytic site are important for the tertiary structure of the ATCase, giving rise to enzyme folding by polar intrachain interactions. Furthermore, amino acids which have been identified i_n E coli and B subtilis ATCase to be involved in interchain associations within the catalytic trimer [39] were also found in the L leichmannii enzyme, pointing to a similar structure. Regions important for the interaction with regulatory polypeptides found in E coil are obviously not well conserved in L leichmannii. Hydrophobicity analysis [40] yields a factor of -0.32 implying a weakly hydrophilic, not membranebound, soluble nature for the pyrB-encoded protein with no predominating hydrophobic regions. Analysis of the amino acid composition revealed furthermore an isoelectric point of the pyrB gene product of pH 5.08. The codon usage bias determined as a P2 value of 0.623 according to Gouy and Gautier [41] points to an efficiently expressed gene. Transcription of the pyrB gene The mapping of the 5' ends of pyrB-mRNA isolated from L leichmanmi and E coil YA289(pJB7) showed that transcription is initiated in either c~se 54 bp upstream of the putative ATG start codon (fig 5). These results confirmed that the pyrB gene is transcribed in E coil by its own promoter and not by a promoter of the vector pBluescript. Knowing the transcription initiation point, the -35 and -10 regions of the promoter can be assumed. Northern blot analysis confirmed the length of the pyrB ORF and presented no indication that the overlapping pyrB and pyrC genes were transcribed as a bicistronic mRNA which must have a length of at least 2.4 kb (fig 6). This result was also obtained very clearly by analyzing total RNA from L leichmannii and the pyrC-deficient E coli strain MAI008 transformed with plasmid pJB9 carrying the entire pyrC gene o f L leichmannii [16]. The pyrB ORF is followed by an inverted repeat which can form a hairpin in the transcript with an estimated free energy o f - 7 . 9 kcal/ mol. Enzymatic activity The ATCase enzyme activities of cell extracts of L leichmannii, E coli YA289(pJB6), E coli YA289(pJB7),

E coli YA289(pJB 11 a), E coil YA289(pJB 111 ), E coli YA289(pBluescript), E coli YA289(pBR322), and E coli YA289(pCK19) were tested under the standard assay conditions (E coli transformed with the empty vectors served as controls). ATCase activity was only measured in cell extracts of L !eichmannii (grown in MRS) and E coli YA289(pJBlll) grown in M9 minimal medium without uracil. No enzymatic activities were detected in E coli transformed with plasmids pJB6, pJB7, pJBlla, pBluescript, pBR322, pCK19, and in E coli YA289(pJB111) grown in LB or M9 minimal medium with uracil (table II). Compared with L leichmannii, ATCase activity in E coli YA289( p J B l l l ) cell extract was approximately 3.2-fold lower. Discussion

Unlike the pyrC gene [16], the entire pyrB gene of L leichmannii could not be cloned via comp'..-mentatk~n of the corresponding E coil pyrB-mutant. However, sequencing of a DNA fragment carrying the entire pyrC gene revealed the C-terminal part of the pyrB ORF [ 16] which offered the opportunJ',~y to clone the lacking N'-terminal part of the L leichmaJmii pyrB gene by screening the genomic library via colony hybridization. Although we could extend the pyrB ORF to some extent, all attempts failed to clone the complete gene including the promoter region. By using newly constructed partial L lewhmannii genomic libraries, we expected to find a number of clones canying the entire pyrB gene but actually only one positive clone could be isolated from a partial Pstl/BstXl library. After subcloning and sequence determination of the resulting plasmid pJB7 we found that it contained the complete pyrB gene, apparently destroyed by a frame shift mutation resulting presumably in an inactive polypeptide. Cloning of a DNAfragment synthesized via PCR with chromosomal L leichmannii DNA as template covering this critical region proved the accuracy of our assumption and indicated that cloning of the entire pyrB 'wild type' gene of L leichmannii on a multi-copy plasmid may not be possible because overexpressed ATCase is toxic. Toxicity of enhanced ATCase activity, presumably caused by an overproduction and accumulation of the reaction end product carbamyl aspartate, was shown by Tumbough and Bochner [42] for Salmonella typhimurium. Therefore, we ligated the defect pyrB gene together with the entire pyrC gene into pBR322 that has a clearly lower copy number and then replaced the frame shift mutation with the correct DNA sequence. E coli pyrB mutants transformed with the newly constructed plasmid pJBlla were not growth-impaired on LB medium or minimal medium

F~g 4. sequence

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medium without uracil probably leads to derepression of the pyrB gene and enhanced ATCase levels; therefore, survival of the host cells is only possible when the gene is present in few copies (3-5 copies/cell). These E coli cells (YA289[pJBllI]), which grow, although very slowly, even under derepressed conditions at uracil starvation, exhibit ATCase activity. The relative low ATCase activity is perhaps due to the low copy number of the plasmid and a weak expression of the pyrB gene (RNA from these cells gave only very weak signals in Northern blotting; data not shown). Whether the pyrB gene of L leichmannii is in fact controlled by UTP as in E coil [43-45] or S typhimu-

123456

3,2 ~ Fig 5. Primer extension analysis of the 5' termini of the pyrB transcripts of L ieichmannii and E coli YA289(pJB7) resp YA289(pJBII). A synthetic oligonucleotide primer complementary to nucleotides 380 to 400 was annealed to total RNA and extended with AMV reverse transcriptase. An autoradiogram of the 6% polyacrylamide sequencing gel used to analyze the primer extension products is shown: lane I and 4, total cellular L leichmam~ii RNA; lane 2, fetal cellular E coli YA289(pJB7) RNA; l,'me 3, total cellular E coil YA289(pJB! la) RNA. The GATC sequencing kmes were generated with the same oligonucleotide primer that was used in the primer extension reaction. The arrow indicates the size of the complete primer extension product, ie the start point of transcription.

with uracil. However, growth on minimal medium without uracil was not possible. The fact that the entire pyrB 'wild type' gene together with the pyrC gene in a lower copy number (15-20 copies/cell) confers the ability to grow on uracil-containing medium to its pyrB- host (which hereby shows no detectable ATCase activity in the cell extracts; data not shown) but not on minimal medium without uracil, indicates that the gene may be repressed by uracil or its derivatives (probably UTP), thus permitting survival. In a high-copy vector like pBluescript with up to 700 copies/cell this repression is not sufficient to prevent a lethal effect. Growth in minimal

1.5

3,2 . 1.5

Fig 6. Northern blot analysis of pyrB transcripts. 2 lag RNA samples were treated with formaldehyde and formamide, electrophoresed on a I C/~ formaklehyde-agarose gel, transfen'ed to nitrocellulose filters and probed with the 536 bp Ncol/Apal fragment of pJB7. Lane 3 contains L leichmannii RNA, lanes 2 and 5 RNA isolated from E coli YA289(pJB7) resp E coli YX289(pJBll) grown in uracil-supplemented medium and lane 4, as negative control, E coli MAI008(pJB9) RNA. The size of the identified RNAs is approximately i.2 kb as deduced by comparison with E coli rRNA size standards (lanes 1 zlnd 6). The fainter upper bands are most probably caused by renatured RNA, the fainter lower bands by degrada!ion products as deduced from other Northern blotting exp~riments (data not shown). The migration of the 1.5 and 3. ~. kb size markers is indicatcd by arrows.

rium [46, 47] has still to be elucidated. Hutson and Downing 1711 observed a four- to five-fold decrease in the concentration of t~spartate transcarbamylase when L leichmanJ~ii-cells were grown in media with uracil. However, these authors could not detect any effects on the activity of the ATCase by the four common ribonucleoside-5'-mono- and triphosphates. These results agree well with our findings that UTR CTR UMP or CMP have no effects on the ATCase activity (data not shown). The deduced amino acid sequence shows censidcrable identities towards ATCases of various other organisms, especially to that of B subtilis. Some highly conserved regions give strong indication about the catalytic sites of the enzyme lying to a large extent in the N'-terminal (polar) doraain where the conserved sequence from Phe-81 to (3 lu-93 is thought to be responsible for carbamyl pi~osphate binding and is typical for carbamoyltrans~erases. The residues Ser85, Arg-87, Thr-88 and perhaps Thr-90 resp Glu-93 are most probably implicat;~d in binding to the phosphoryl group of carbam,,l phosphate [39]. Other amino acid residues that also seem to be involved in carbamyl phosphate binding (eg Arg-137 and His170) are also well conserved in the presented ATCases and OTCases, indicating that these enzymes descend from a common ancestral transcarbamylase [48]. However, ATCase residues involved in binding to the [3-carboxylate of aspartate (eg Arg-257 and Gin-259) are absent in OTCases. Homologous regions characteristic for intrachain interactions and interchain associations as within the E coli catalytic trimer could be detected. On the other hand the region in the polar domain of the E coil ATCase which forms part of the interface between the catalytic and regulatory subunits is not well conserved in the L leichmmmii enzyme with the exception of Ser-43 and Arg-149; the latter amino acid seems to be i~volved in allosteric interactions in the E coil enzyme. This indicates that the L ieichmannii ATCase like that of B subtilis [49] could exist as a catalytic polymer, perhaps a trimer, and is probably not linked to regulatory subunits like the E coil or S typhimurium enzyme [9, 47, 501. The promoter region could be deduced by determining the 5' end of the RNA transcript via primer extension. The -35 region is in good agreement with the known consensus sequence of prokaryotes but the - 1 0 region differs at some positions from the E coli ¢J70 or B subtilis c~43 promoter-like consensus sequences, a fact that is not unusual for Lactobacillus promoters [51]. The putative ribosomal binding site (Shine-Dalgarno-sequence) agrees also well with the known consensus motif, and the codon usage at least indicates a good translation of the transcribed gene. In contrast to the structural pyrB genes of E coli and S typhimurium there are no other transcriptional control

sequences preceding the pyrB ORF of L h'ichntattnii, e.- neither regions of dyad symmetry permitting the formation of transcription-terminating RNA hairpins nor pyrimidine-rich regions causing the RNA polymerase to pause at low levels of UTP nor smalt ORFs encoding putative leader peptides. Thus, we can conch, de that the L leichmannii pyrB gene is not regulated by an UTP-sensitive attenuation mechanism which controls, dependent on the cellular UTP pool, transcriptional readthrough into the structural genes as postulated for pyrB transcriptional regulation in E co/i and S O'phimurium [12, 52-55]. An attenuator mechanism controlling the pyr operons of B subtilis and B caldolyticus [56, 57] with putative transcription terminators resembling E coli rho-indepenaent terminators and antiterminators with conserved sequences for pyrimidine nucleotide-depending binding of the regulatory pyrR protein could also not be identified. The regulation of the L leichmanni pyrB gone might be different from that of B subtilis, E coli and other organisms considering our finding that a high gene copy number is lethal to E coli, a fact not reported for other pyrB genes. The pyrB ORF of L leichmannii is followed by three stop codons and a region of dyad symmetry which is capable of forming a secondary hairpin structure but seems not to be a transcription termination signal. Independent transcription of the gone could be confirmed by Northern blot analysis which revealed no clear signal that would correspond to a bicistronic mRNA comprising the two ORFs. This indicates that the pyrB gone is not transcribed together with the following overlapping pyrC gene (that is in fac~ transcribed bS/ i~s own promoter [16]) as a bicis~ronic mRNA from a common promoter as might be proposed by considering that an overlapping of star~ and stop codons of sequentially expressed genes in operons provide a mean for the translational coupling of expression [58]. Nevertheless, a possible growth condition-dependent linkage of expression of the two pyr genes leading to stoichiometric production of the two polypeptides cannot be excluded entirely. In the evolution of the pyr genes, L leichmannii seems to have an intermediary position between the Gramnegative prokaryotes and B subtilis resp B caldolytitus.

Acknowledgmenls We wish to thank M Markovic for helping to draw the pictures and Barbara M Bachrnann, Kai F Jensen, and the BGSC for donation of bacterial strains. Special thanks to the groups of A Kr6ger and KD Entian for making available plasmids pCKI9 and pFIVI432 and the use of the DNASIS and PROSIS program. Data are from the doctoral thesis of JB who held fellowships from the Fonds der Chemischen Industrie and the Graduiertenf6rderung des Landes Hessen.

12

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