Genes and Enzymes of the Acetyl Cycle of Arginine Bio Synthesis in C. cum Enzyme Evolution in the...

8/2/2019 Genes and Enzymes of the Acetyl Cycle of Arginine Bio Synthesis in C. cum Enzyme Evolution in the Early Steps of

1/10

Microbiology (1996), 142, 99-1 08 Printed in Great Britain

Genes and enzymes of the acetyl cycle ofarg n ne biosy n hesis in Corynebacteriumglutamicum: enzyme evolution in the earlysteps of the arginine pathwayVehary Sakanyan, Pave1 Petrosyan, Mich ele Lecocq, A nn e B ~ y e n , ~ . ~Chri st iane Leg ra i r~ ,~arc Demarez, Jea n-No d Hal let an dN coIas GIansdorf 14rAuthor for correspondence : Vehary Sakanyan. Tel : +33 40373065. Fax: +33 40292042.

1 Laboratoire deBiotechnologie, FacultCdes Sciences et desTechniques, Un versi C deNantes, 2, rue de laHoussin We, F-44072Nantes Cedex 03, FrancePharmagen, KnunyantsStreet 4, Yerevan 375010,Republicof Armenia

3,415 Microbiologie, VrijeUniversiteit Brussel3,Vlaams lnteruniversitairInstituut voorBiotechnologie4, ResearchInstitute, CERIA-COOVI5,1 avenue Emile Gryson,B-1070 Brussels, Belgium

A cluster of arginine biosynthetic genesof Corynebacterium glutamicumATCC 13032,comprising argJ, argB and argD as we ll as part of argC and argF,has been cloned by heterologous complementation of an Escherichia coli argEmutant. The gene order has been established as argUBDF by sequencing theentire 4 4 kb cloned DNA fragment. The C glutamicum argB gene can betranscribed in E. coli cells from an internal promoter located in th e coding partof t he preceding argJ gene, whereas transcription of th e argl gene appearsvector-dependent. Expressionof the corynebacterial argB gene i s repressed byarginine in the native host but not in recombinant E. colicells. Feedbackinhibition of the corresponding N-acetylglutamate kinase activity wasobserved both in cell extracts of C. glutamicum and in recombinant E. coliargBauxotrophic strains. Extracts of E. colicells carrying cloned corynebacterialDNA display an orn ithine acetyltransferase activity (encoded by argJ)whichalleviates the acetylornithinase (encoded by argq deficiency of th een erobacter aI ost. In contrast to Bacillus stearothermophilus orni h eacetyltransferase which also exhibits acetylglutamate synthase activity, C.glutamicum orni thine acetyltransferase appears monofunctional. ArgA andArgB proteins from dif ferent sources share hig hly significant similarities. Theevolutionary implications of these data are discussed.

Keywords: Coymebacterium glutamicum, argCJBDF sequence, ornithine acetyltransferase,N-acetylglutamate kinase, regulation

INTRODUCTIONArginine biosynthesis sets off with the acetylation of theamino group of glutamate (Fig.l), mediated by N-acetylglutamate synthase (acetylCoA :L-glutamate N-acetyltransferase; EC 2 .3. 1 . l ; he a r g A gene product).This acetylation prevents spontaneous cyclization andhence proline formation during the subsequent modi-fications of the 5-carboxyl group. Two strategies haveevolved for the ulterior removal of the acetyl group. Inthe so-called linear pathway operative in Enterobacteriaceaeand in the archaeon Stllfolobtlssolfatarictls acetylornithinase(N2-acetyl-L-ornithine amidohydrolase; EC 3.5.1 . 16 ;the argE gene product) catalyses the hydrolysis of N-. . . . . . . . . . . . . . . . .The EMBL accession n umbe r for the sequence reported in this paper isX86157.

acetylornithine into the arginine precursor ornithine andacetate (Cunin e t al., 1986; Van de Casteele e t al., 1990).All other prokaryotes investigated up to now, includingmethanogenic archaea (Meile & Leisinger, 1984; Van deCasteele e t al., 1990), Neisseria gonorrhoeae (Shinners &Catlin, 1978), members of the genus Bacilltls (Sakanyane t al., 1992) and the eukaryotic microbes (Davis, 1986) usethe more economical cyclic pathway that was first broughtto light in Micrococctls gltltamictls (Udaka & Kinoshita,1958), in which the acetyl group is recycled withgeneration of acetylglutamate. The transacetylationbetween acetylornithine and glutamate is mediated bythe aryJ gene product, ornithine acetyltransferase (N2 -acetyl-L-ornithine :L-glutamate N-acetyltransferase ; EC2.3.1.35). In organisms depending on the cyclic path-way, N-acetylglutamate synthase therefore fulfills ananaplero ic function.

0002-0252 0 1996 SG M 99


2/10

V. S A K A N Y A N a n d O T H E R S

argAGlutamate

Glutamylphosphate

Glutamate semialdehyde

Pyrroline-5carboxylate

Proline

HC03-

NAcetylglutamateIII argBI I C A T D P

I NAcetylglutamyl phosphateI NADPH+II J NADP +PiI NAcetylglutamateI sem aldehydeIII

II Mcetylornithlne

erg2 A c e t a t eOrnlthine

ATP + arbamoylphosphateGlutamine argFCitrulline

argGF:;PIArgininosucclnate

argHFumarateArginine

Fig. 1. Arginine biosynthesis and i tsrelationship to the proline pathway. Thedashed line indicates the acetyl grouprecycling by the arg/-encoded enzyme as analternative to deacetylation by the argEgene product.

Complementation experiments with N. gonorrhoeae (Picard& Dillon, 1989; Martin & Mulks, 1992) and Bacillztsstearothermophilztx (Sakanyan e t al., 1990) revealed that onesmall DNA fragment of these organisms could comp-lement both a r g A and argE auxotrophs in E. coli. Theresponsible genes have since been sequenced and sufficientgenetic (Martin & Mulks, 1992; Sakanyan etal., 1992) andenzymic (Sakanyan e t al., 1993a) data have been accumu-lated to prove that these organisms possess a bifunctionalargf-encoded product capable of using both the acetylgroup of N-acetylornithine and that of acetylCoA toacetylate glutamate. It appears that Bacillztx sztbtilis alsoharbours a bifunctional acetyltransferase (OReilly &Devine, 1994).Although the arg/ gene by itself would thus be able toassure both the first and the fifth steps of argininebiosynthesis in these organisms, there is genetic evidencefor the existence of an independent functional a r g A genein N. gonorrhoeae (Picard & Dillon, 1989; Martin &Mulks, 1992) and enzyme data for B. stearothermophilztspoint in the same direction (Sakanyan e t al., 1992). In

Pseztuomonas aerztginosa, however, the synthase and trans-acetylase activities can be separated by gel filtration (Haase t al., 1972). Moreover, a r g A mutants have been isolatedwhich display a normal acetyltransferase but no synthaseactivity and hence an arginineless phenotype. It has beenshown that the synthase enzymes of P. aerztginosa (Haas e tal., 1972; Haas & Leisinger, 1974) and Saccharomyescerevisiae (Wipf & Leisinger, 1979) lack orni thine acetyl-transferase activity ; the properties of the ornithineacetyltransferases were not studied in detail. The data forP. aerztginosa therefore suggest the existence of a mono-functional ornithine acetyltransferase in this organism.Similarly, the cloned Streptomyes coel icolor ornithineacetyltransferase gene complements E. coli argE but nota r g A mutants (Hindle e t al., 1994).In some micro-organisms the metabolic flow through theacetyl cycle is controlled by arginine-mediated feedbackinhibition of the second biosynthetic step, catalysed byN-acetylglutamate kinase (ATP :N-acetyl-L-glutamate 5-phosphotransferase ;EC 2 .7 .2 .8 ;Udaka, 1966;Hoare &Hoare, 1966;Haas & Leisinger, 1975;Meile & Leisinger,

100


3/10

Arginine biosynthesis in CorJnebacteriumglutamicumTable 1. Bacterial strains and plasmids used in this study

Strainlplasmid Relevant genotypelphenotype RefetencelsourceE. coli K12JM109

XA4XB25xc33PT2XFXG31Xl90XJEF8XSlD2RX A 4 a r g EC. lutamicumATCC 13032PlasmidspBR327pUCl8pGA46pBluescript I1 KS( +)

puc9

pPP2

e14- (McrA-) recA 1 endA 1g y r A 9 6 thi- 1hsdR 17 szlpE44 relA 1 A(lac-proAB)[F' t raD 36p roAB lacIqZAM 151

F- argA na lA I - I" h sdRF- argB nal A I - I s hsdRF- argC n alA rpoB I+ hsdRF- argD pr oA B his i l vA metB rpsLF- A101 (proAB-argF-lac) argI th i supEHfr argG his rpoB rpsL 1- 1 hsdRHfr(P4X) ca rA metB thr I+ W202: :TnlOHfr(P4X) carB metB thr I+ uj202: :TnlOF- A(ppc-argE) na lA rpoB I - hsdR recAAs XA4, but argE86: :Tn 10Wild-type

I - As hsdR hsdM

hsdRhsdR

Ap' Tc'Ap' lacZAp' lacZAp' Cm'Ap' lacZpBR327-derivative, Ap' Tc'

Yanisch-Perron et al. (1985)

Mountain e t al . (1984)Mountain e t al . (1984)Mountain e t al. (1984)Mountain e t al. (1984)Mountain e t al . (1984)Mountain et al . (1984)Mountain et al. (1984)Mountain e t al . (1984)Sakanyan e t al. (1992)Sakanyan e t al . (1992)Collection of IndustrialMicroorganisms, Moscow,Russia

Bolivar (1978)Vieira & Messing (1984)Yanisch-Perron e t al . (1985)An & Friesen (1979)S ratageneThis work

1984). However, in B. stearothermophilzrs no noticeableinhibition of N-acetylglutamate kinase by either ornithi neor arginine could be detected. Instead, the target forinhibition was found to be the bifunctional a r d (andpossibly the a r g A ) gene product : bot h N-acetylglutamatesynthase and ornithine acetyltransferase activities werestrongly inhibited by ornithine. Arginine, however, didnot affect either activity (Sakanyan e t al., 1993a). Conse-quently, in this organism the metabolic intermediateornithine, rather than the end-pro duct arginine appears tobe critical for controlling metabolite conversions in thearginine acetyl cycle.In o rder t o clarify the organization and the regulation ofthe cyclic acetylation pathway a more extensive study wasdesirable. W e have un dertaken t he genetic and enzymicexamination of the pathway in Cor_ynebacterizrm hta micu m,formerly Micrococcztsglzrtamicm (Jones & Collins, 1986), aGram-positive mesophilic bacterium. Indigenous non-pathogenic corynebacteria, particularly representatives ofCorJynebacterizrman d Brevibacterizrm, synthesize and excretelarge quantities of glutamic acid in b roth cultures (Shiio e tal., 1962). Genetically improved strains have thereforelong been exploited for the industrial production ofarginine and proline (Kinoshita & Nahayama, 1978), butthe underlying genetics has not been studied extensively.The data reported below provide new information on theacetyl cycle in this org anis m.

METHODSBacterial strains and plasmids. The strains and plasmids usedin this work are listed in Table 1.Media and growth conditions. C.glutamicum cells were grownon a rotary shaker (150 r.p.m.) at 30 "C in Luria-Bertani (LB)medium (Sambrook e t al., 1989) or in a synthetic mediumdescribed by Broer e t a/ . (1993). LB as well as the synthetic M9medium (Miller, 1972) were used for E. coli K12 strains. M9 wassupplemented with all auxotrophic requirements other thanarginine for complementation analysis of arg mutants by C.glutamicum DNA. E . coli cells were grown at 37 OC, except forcomplementation tests which were performed at 30 OC. Anti-biotics were added at a final concentration of 50 pg ml-' forampicillin, 30 pg ml-' for chloramphenicol and 25 pg ml-' fortetracycline.DNA manipulation and transformation. Chromosomal DNAof C. glutamictlm was isolated as described by Eikmanns e t al .(1991). Plasmid DN A from E. coli was isolated by the alkalinelysis method of Birnboim & Doly (1979) or with Qiagen-tipcolumns (QIAGEN). Agarose gel electrophoresis, DNA re-striction, alkaline phosphatase treatment and ligation wereperformed following classical protocols (Sambrook et al., 1989).E. coli strains were transformed following the CaCl, procedure(Sambrook e t al., 1989) or by electroporation with thegenezapper 450-2500 apparatus (International Biotech-nologies) according to the manufacturer's recommendations.DNA sequencing and sequence analysis. Prior to sequencingsubfragments were cloned into the pBluescript I1KS(+ vector.

101


4/10


ExoIII-generated unidirectional deletions were obtained ac-cording to the method of Sambrook e t al . (1989). Sequencingwas performed by th e dideoxynucleotide chain-terminationmethod (Sanger e t al., 1977) using sequenase Quick-denaturateplasmid sequencing kits (USB) and [LX-~~SI~ATPAmersham).Either universal or reverse primers or synthetic 17-mer oligo-nucleotides (provided by D. Gigot, Research Institute, CERIA-COOVI, Brussels, and by Eurogentec) were used. Sequenceladders were resolved on gels containing 6 % ( w / v ) poly-acry lamide with taurine. The sequence data were compiled,analysed and aligned with various programs from the Mac-Vector package (InternationalBiotechnologies),as well as withCLUSTAL v (Higgins & Sharp, 1988) and FASTA (Pearson &Lipman, 1988). The Swiss-Prot database (EMBL)was consultedfor protein sequences.Enzyme assays. E . coli and C. glzrtamicum cells were grown insynthetic media, harvested by centrifugation during theexponential phase, washed in 0.9YOw/v) NaCl and resuspendedin 10m M potassium phosphate buffer (pH 6.5) containing 15YO(v/v) glycerol, 1 m M EDTA, 1 mM DTT and 2 m M PMSF forN-acetylglutamate synthase and ornithine acetyltransferaseassays, or in 25 m M Tris/HCl buffer (pH 7.5) fo r other assays.Cells were disrupted by sonication and the resulting crudeextract was centrifuged (20000g,15 min). Al l these treatmentswere performed a t temperatures below 10 "C. Enzyme assayswere carried ou t at 30 "C. For inhibition experiments the crudecell extracts were passed through Sephadex G-25 columnsequilibratedwith extraction buffer.N-Acetylglutamate synthase and ornithine acetyltransferasewere assayed as described in Van de Casteele e t al . (1990), exceptthat 15Yo ( v /v ) glycerol, 10 mM MgCl,, and 7 mM amino-oxyacetic acid were added to th e incubation mixture. N-Acetylglutamate kinase was measured by th e ferric chloridemethod (Udaka, 1966) as described previously by Van deCasteele e t al . (1990). Acetylornithinase was measured by themethod of Vogel & McLellan (1970) as described in Sakanyane t a/. (1993~).RESULTS AND DISCUSSIONCloning of C. glutamicum arginine biosyntheticgenes and their expression in E. coli mutantsCloning of the C.glzttamicztm arg/ gene was undertaken byselecting for heterologous complementation of argEdeficiency in E. co l i as obtained with N. gonorrhoeae and B.stearothermophilzts DNA (Picard & Dilon, 1989;Sakanyane t a/., 1990). An EcoRI-digest of C. glzttamicztm ATCC13032 DNA was ligated into EcoRI-cleaved vectorpBR327. The resulting plasmids were transformed intoE. coli K12 XSlD2R and arginine prototrophs wereselected o n synthetic medium supplemented withsuccinate, ampicillin and tetracycline. The recombinantpPP2 plasmid which carries a single 4.4 kb EcoRI insertwas isolated from the selected transformants. Its restric-tion map derived from single- and double digest datawith several enzymes is shown in Fig. 2.Ampicillin-resistant pPP2 transformants of various ar-ginine auxotrophic E. coli K12 mutants were screened forcomplementation by replica plating on synthetic mediumwithout arginine. Apart from the argE-deficient strainmentioned above, pPP2 complemented argB mutantXB25. Of particular interest to our study is its failure tocomplement argrl-deficient strains XA4 and it s derivative

XA4argE. Enzyme assays summarized in Table 2confirmed these results. N-Acetylglutamate kinase andornithine acetyltransferase activities measured in cellextracts of E. coli K12 XB25(pPP2) and E. coliXA4(pPP2), respectively, were raised significantly abovethe background level of the plasmidless strain. Acetyl-glutamate synthase and acetylornithinase activities, incontrast, remained undetectable in extracts of E. co l i K12XA4(pPP2) and E. co l i K12 XSlD2R(pPP2). From theseresults it can be inferred that the cloned fragment harboursthe structural C. glzrtamiczrm argB and arg/ genes and thatthe argf-encoded ornithine acetyltransferase relieves theacetylornithinase deficiency of the E. co l i argE mutant byheterologous complementation.Since the native C. glzrtamiczrm ATCC 13032 displaysN-acetylglutamate synthase activity (see Table 2) it can beassumed that the corresponding argA gene is not locatedon the pPP2 plasmid unless it is non-functional in E. colicells. Consequently, the cloned C. glzttamiczim arg/ geneappears to encode a monofunctional enzyme capable oftransacetylating the acetyl group of N-acetylornithine(orn ithine acetyltransferase activity), but not that ofacetylCoA (N-acetylglutamate synthase activity) toglutamate.

Sequence analysisThe sequence of the entire 4.4 kb insert of C.glzitamiczrmDNA in pPP2 has been established o n both strands usingExoIII-generated 200-300 bp deletions in a subcloned1.9 kb HindIII-XhoI fragment (plasmid pKS-1.9) andseveral other subcloned fragments. Analysis of thenucleotide sequence reveals five large ORFs (Fig. 3)oriented in the same way, those at the fragments' endsbeing truncated. The ORFs were numbered as indicatedin Fig. 2. Comparison of the corresponding amino acidsequences with known arginine metabolic enzymes fromvarious sources (see also below) shows that all of themcorrespond to arginine biosynthetic genes : truncatedORFl matches the C-terminal end of the argC-encodedpolypeptide, O RF 2,OR F3 and ORF4 appear respectivelyhomologous with the arg/-, B- and D-encoded enzymesand finally, the truncated O RF5 corresponds with the N-terminal region of the argF gene product. Putative RBSscan be found upstream of the proposed initiation codonsof ORFs 2-5 and are indicated in Fig. 3. The cloned 4.4 kbinsert therefore seems to contain an important part of alarge arginine biosynthetic cluster as in B. szrbtilis (Moun-tain e t al., 1986), N. onorrhoeae (Picard & Dilon, 1989),B. stearothermophilzrs (Sakanyan e t al., 1990 ;Sakanyan e t al.,1993a) and S. oelicolor (Hindle e t al., 1994). A fairly largeORF6 oriented in the opposite direction showed nosignificant resemblance to any sequence as yet regis-tered in the protein databases, Searching specifically forhomology between C. glzrtamiczrm ORFs and the knownacetylglutamate synthase sequences of E. coli (Brown e tal., 1987), P. aerzrginosa and Psezrdomonas pzrtida(Dharmsthiti& Krishnapillai, 1993)and Neztrospora crassa(Y. Yu & R. L. Weiss, unpublished; EMBL accessionnumber L35484) was negative, except for ORF3 which

102


5/10

Arginine biosynthesis in Coynebacterinmglntamicnm

< ORF6 I00- I ORF2 > ORF3 3 ORF4 >I ORF5 innn

RI. . .d 1do0argC argJ 2000argB 3000argD 4000 4355 bpargF

Com plementat on:tOrnithinet t t tN-Acetylg utamate N-Acety o nith ne-Acetylglutamate Ornithinesemialdehyde acetyltransferase kinase aminotransferase carbam oyltransferase argE argBdehyd rogenasepPP2 + +pEX4 - +pGAB4 - +PKS-1.9 - +

............................................................... . ............. ............................... ..... ............................................ .................................................... ..... ... ......... ........ ... .... ... .... .... ............ .................................... ..... ...Fig. 2. Genetic and restriction maps of th e 4.4 kb insert of C. glutarnicurn DNA in pPP2. The arrowhead boxes show thelocalization and o rientation of th e ORFs. The corresponding genes and th e encoded enzymes are indic ated. Plasmid pEX4was constructed by cloning the EcoRI-Xhol frag ment from pPP2 into EcoRIISall-digested pUC9, and plasmid pGAB4 wasconstructed by cloning the Hindlll-Xho l frag ment into HindIIl/Sall-digested pGA 46. These plasmids wer e used incomplementation experiments with E. coli K12 XB25 and XSlD2R strains (results indicated to th e right). The Hindlll-Xholfragment from pPP2 was also cloned into the HindlllISall sites of pUC18 and re-cloned after excision with Hindlll andEcoRl (takin g advan tage of the polylinker site) into HindllIIEcoRI-digested pBluescript II KS(+), yielding plasmid pKS-1.9.

corresponds to the argB gene. This homology betweenacetylglutamate synthase and acetylglutamate kinase issupported by the comparison of othe r a r g A and argB genecouples (Reith & Munholland, 1993; see below).According to a BESTFIT comparison, the C. glzrtamicnmArgJ sequence shares 36, 35 and 39 YO dentical aminoacids with the o rnithine acetyltransferases of N. onorrboeae(Martin & Mulks, 1992), B. szrbtilis (OReilly & Devine,1994) and B. stearotbermophilns (Sakanyan e t al ., 1993a),respectively. The CLUSTAL alignment shown in Fig. 4indicates that the similarity covers the whole sequence,though the C. glntamicnm polypeptide appears shorterthan its bifunctional homo logues by 11-12 amin o acids atthe N-terminal end. Its predicted molecular mass is39.8 kDa, approximately 3 kDa less than for the oth erknown bacterial ornithine acetyltransferases ; it containsonly 6.7% of basic amino acids which is substantiallylower than the 10 % found for the other ArgJ products.A BESTFIT comparison show s that the N -acetylglutamatekinase polypeptide sequence of C. glntamiczrm sharesapproximately 42% identical amino acids with the N-acetylglutamate kinase of Porphyra nmbilicalis (Reith &Munh olland, 1993), 39 YOwith that of B. stearotbermophilns(Sakanyan e t al., 1993b), 35 % with that of B. snbtilis(OReilly & Devine 1994), 29 % w ith that of E. coli(Parsot etal . , l988), 25 YOwith that of N . crassa (Gessert e tal., 1994) and 23 YOwith those of S. cerevisiae (Boonchird e tal., 1991) and Scbi~osaccbarom_ycespombeVan Huffel e t al.,1992).Sequence analysis indicates that the initiation c odo n of theC. glzrtamicnm argD gene is contiguous to the argB T A Atermination codon, whereas an intergenic space is

observed between the other corynebacterial arg genes ofthe cluster. The same tendency was observed in B.stearothermophilm (Sakanyan e t al., 1993b) and B. szrbtilis(OReilly & Devine, 1994), where the argB an d argDgenes overlap for a few nucleotides and suggests trans-lational coupling, while a relatively long intergenic spacewas found at the argC/ard an d argJlargB transitions. ACLUSTAL alignment of the deduced ArgD polypeptidesequence with those of E. coli , B. snbtilis and S. erevisiae(data not sh own) revealed 40 ,42 and 36 % identical am inoacids, respectively.T h e o v er al l G + C co n ten t o f t he 4 4 k b s tr et ch o f C.glzrtamiczrm D N A a m o u nt s to 54.6 Y O ,which correspondswith the mean value for glutamic-acid-producingcorynebacteria (Yamada & Komagata, 1970). A remark-able feature in codon usage is the unexpectedly highappearance of the rare CGA arginine codon in the arg/gene (4 ou t of 12 codons). A part fro m that there is arelatively low occurrence of G +C in the third position ofth e a r - odons: 51.7 YO s compared to 59.3 and 64.1 %for the argB an d argD genes, respectively. There is nostrong bias for corynebacterial preferred codons(Malumbres e t al., 1993) in either a r d , argB o r argD;therefore a low-to-moderate expression might beexpected.Evidence for a secondary promoter upstream of theargl3 geneFrom the sequence analysis it appears that the pEX4plasmid, cons tructed by subclo ning the 3.1 kb EcoRI-XboI fragment in EcoRIISalI-cut pUC9 vector, shouldcarry the whole of the structural information for both th e

103


6/10

V. S A K A N Y A N a n d OTHERS

argCI L T T A T A P L K E G V T A E Q A R

GAUETCACCACTGCAACCGCACC"GAAAGAAGGCGTTACCGCAGAACAGGCTCGCGA V Y E E F Y A Q E T F V H V L P E G ACAGTATATGAAGAGTTCTATGCACAGGAAACCTTCGTGCATGTTCTTCCAGAAGGTGCACQ P Q T Q A V L G S N M C H V Q V E I DAGCCACAAACCCAAGCAGTTCTCTTGGCTCCAACATGTGCCACGTGCAGGTAGAMTTGATGE E A G K V L V T S A I D N L T K G T AAGGAAGCAGGCAAAGTCCTTACCWCGCAATCGATAACCTCACCAA~CTGCCGG A A V Q C M N L S V G F D E A A G L PGCGCCGCTGTTCAGTGCATGAACTTAAGCGTTGGTTTEA'TGAGGCAGCAGGCCTGCCAC

Q V G V A P *AGGTCGGCGTCGCACCTTARAGTAGCGCCTTAAAGCGGCGCTTCAAACCAAGCGCCCTAAM A E K G I

C C A GC A A A C A C A A C R A A C A C A T C T A A T T C A GT A GG& IT ' T C C A C A T GGC A GU ~ A TT A P K G F V A S A T T A G I K A S G NT A C C G C G C C G A A A G G C T T C G T ' I G C T ' T C T G C A A C G A C C G C G G G T A T l ' m m A AP D M A L V V N Q G P E F S A A A V F TTCCTGACATGGCGTTGGTGGTTAACCAGGGTCCAGAGTTTTCCGCAGCGGCCGTGTTTACR N R V F A A P V K V S R E N V A D G QACGTAACCGAGmTCGCAGCGCCTGTGAAGGTGAGCCGAGAGAACGTTGCTGAmCAI R A V L Y N A G N A N A C N G L Q G EG A T C A G G G C T G m T G T A C A A C G C T G G T ~ ~ C T A A T G C G T G T A A T G G T ~ G G T G AK D A R E S V S H L A Q N L G L E D S D

GAAGGATGCTCGTGAGTCTGTl"CTCAWTAGCWAAAAl"rTGGGC'ITGGAGGAmCGAI G V C S T G L I G E L L P M D K L N ATA?TGGTGTGTGTTCCACTGGWTTAmGTGAGTTGCTWCGATGGATAAGCTCAATGCG I D Q L T A E A P L G D N G A A A A KAGGTATTGATCAGCTGACCGCTGAGGCACCTTTGGGTGACAATGGTGCAGCTGCTGC~A I M T T D T V D K E T V V F A D G W T

GGCGATCATGACCACTGACACGGCACGGTGGATAAGGAAACCGTCGTGTMGCIGATGGTTGGACV G G M G K G V G M M A P S L A T M L V

T G T C G G C G G A A T G G G C A ~ A ~ T G G C G C C G T C T C T ' I G C C A C C A T G C ~ TC L T T D A S V T Q E M A Q I A L A N A

CTGCTTGACCACTGATGCATCCGTTACWAGGAAATGGCTCAGATCGCGCTGGCTAATGCT A V T F D T L D I D G S T S T N D T V

TACGGCCGTTACGTTTGACACCCTGGATATTGATGGATCAACCWCACCAATGACACCGTF L L A S G A S G I T P T Q D E . L N D A

GTTCCTGCTGGCATCTGGCGCTAGCGGAATCACCCCAACTCAGGATGAACTCAACGATGCV Y A A C S D I A A K L Q A D A E G V TGGTGTACGCAGCTTGTTCGCAGCGAAGCTZCAGGCTGATGCAGAGGGTGTGACK R V A V T V V G T T N N E Q A I N A A

C A A G C G C G T T G C T G T G A C A G T G G T G G G A A C C A C C A A C A AR T V A R D N L F K C A M F G S D P N WTCGCACTGTTGCTCGTGACAA~T'TCAAGTGCGCAATG~A~TGATCCAAACTGG R V L A A V G M A D A D M E P E K I S

GGGTCGCGTGTTGG~WGGCT G G C T G A T G C T G A T A T G G A A C C A G A G ~ G A ~V F F N G Q A V C L D S T G A P G A R E

TG TG TTCTTCA A TG G TCG TA TG CCT' TG A T' TCCA Cm G CW CTG G TG CW G TG AV D L S G A D I D V R I D L G T S G E GGGTGGATCTTTCCGGCGCTGACATTGATGTCCGA4TTGA"XGCACCAG~GGQ A T V R T T D L S F S Y V E I N S A Y

CCAGGCAACAGTTCGAACCACTGACCTGAGCTTCKCTACGTGGAGATCAACTCCGCGTAs s * M N

CAGCTCTTAAAAAGAAACAGCACTCCAACTAACAAGCAGGG~ACAGGCATGAAD L I K D L G S E V R A N V L A E A L PT G A C T T G A T C R A A G A T T T A G G C T C T G A G G T G C G C G C G C A A A T CW L Q H F R D K I V V V K Y G G N A M V

ATGGTTGCAGCACTTCCGCGACAAGATTGTTGTCGTGAAATATGGCGGAAACGCCATGGTD D D L K A A F A A D M V F L R T V G AGGATGATGATCTCAAGGCTGCTT?TGCTGCCGACATGGTCT'TC~GCACCGTGGGCGCK P V V V H G G G P Q I S E M L N R V GA A A A C C A G T G G T G G T G C A C G G T G G T G G A C C T C A G A T T T C TL Q G E F K G G F R V T T P E V M D I V

TCTCCAGGGCGAGTTCAAGGGTGGTTTCCGTGTGACCACTCCTGAGGTCATGGACATTGTR M V L F G Q V G R D L V G L I N S H G

G C G C A T G G T G C T C T T T G G T C A G G T C G G T C G C G A T T T A G T TP Y A V G T S G E D A G L F T A Q K R M

CCCTTACGCTGTGGGAACCTCCGGTGAGGATGCCGGCCTGTTTACCGCGCA-GCGCATV N I D G V P T D I G L V G D I I N V DGGTCAAC-GGCGTACCCACTGATATTGGTTTGGTCGGAGACATCATTAATGWGAA S S L M D I I E A G R I P V V S T I A

TGCCTCTTCCTTGATGGATATCAWGAGGCCGGTCGCATTCCTGTGGTCTCTACGATTGCP G E D G Q I Y N I N A D T A A G A L A

TCCAGGCGAAGACGGCCAGATTTACAACATTMCGCCGATACCGCAGCAGG~

. b R I

arg/RBS

nMii

Pia

EmRv Hindlll

Pia

argBRBS

ECORV

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

LO20

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

2040

2100

2160

2220

A A I G A E R L L V L T N V E G L Y T D~CGATTGGTGCAGAACGCCTGCTGGTTCTCACCAATGTGGAAGGTCTGTACACCGA 2280PdW P D K S S L V S K I K A T E L E A I LT T G G C C T G A T A A G A G C T C A C T G G T G T C C A A G A T C A A G G C C T C T 2340P G L D S G M I P K M E S C L N A V R GWCGGGACTTGATTCCGGCATGATTCCAAAGATGGAGTCTTGCTTGAACGCGGTGCGTGG2 00G V S A A H V I D G R I A H S V L L E LGGGAGTAAGCGCTGCTCATGTCATTGACGGCCGCAWGCGCACTCGGTG~CTGGAGCT 2460L T M G G I G T M V L P D V F D R E N Y

T T T G A C C A T G G G T G G A A T T C G A T G G T G C T G C C G G A ? 2520arso

P E G T V F R K D D K D G E L * M S T LTCCTGAAGGCACCG'MTTTAGAAAAGACGACAAGGATGGGGAACTGTAAATGAGCACGCT 2580RBS

E T W P Q V I I N T Y G T P P V E L V SGGAAACTTGGCCACAGGTCATTATTAATACGTACGGCACCCCACCAG"GAGCTGGTGX 2640G K G A T V T D D Q G N V Y I D L L A GCGGCAAGGGCGCAACCGTCACTGATGACCAGGGCAATG~TACATCGA~~GCGGG700I A V N A L G H A H P A I I E A V T N QCATCGCAGTCAACGCGTTGGCCCACGCCCACCCGGCGATCAWGAGGCGGTCACCAACCA760I G Q L G H V S N L F A S R P V V E V AGATCGGCCAAC'TTGGTCACGTCTCMCTTGTTCGCAWCAGGCCCGTCGTCGAGGTCGC 2820E E L I K R F S L D D A T L A A Q T R V

CGAGGAGCTCATCAAGCGTCGC"GACGACGCCACCCWGCCGCGCAX+CCCmT 2880F F C N S G A E A N E A A F K I A R L TT T T C T T C T G C A A C T C G G G C G C C G A A G C T G C C 2940G R S R I L A A V H G F H G R T M G S Ln;GTCGTTCCCGGATTCTGG-T'TCATGG"TCCACGGCCGCACCATGGGTTCCCT 3000PsllA L T G Q P D K R E A F L P M P S G V ECGCGCTGACTGGCCAGCCAGACAAGCGTGAAGCGTTCCTGCCAATGCCAAGCGGTG?"XA 3060F Y P Y G D T D Y L R K M V E T N P T DGT'TCTACCCTTACGGCGACACCGATTACTTGCGCAAAATGGTAGAAACCAACCCAACGGA 120V A A I F L E P I Q G E T G V V P A P E

TG"ECTATCTTCaCCAAWCAGGGTGAAACGGGCGTTGTTCCAGCACCTGA 3180XholG F L K A V R E L C D E Y G I L M I T D

AGGATTCCTCAAGGCAGTGCGCGAGCTGTGCGATGAGTACGGCATCTTGATGATCACCGA3240E V Q T G V G R T G D F F A H Q H D G VTGAAGTCCAGACTGGCGTCCGTACCGGCGA'MTCTTTGCACATCAGCACGATGGCGT 3300V P D V V T M A K G L G G G L P I G A CT G T T C C C G A T G T G G T G A C C A T G G C C M G G G A C ' I T G G C G G 3360L A L R A A E L M T P G K H G T T F G GTTTGGCACTGCGTGCAGCTGAATTGATGACCCCAGGCAAGCACGGCACCACmCGGTGG 3420N P V A C A A A K A V L S V V D D A F CCAACCCAGTTGCTTGTGCAGCTGCCAAGGCAGTGCTGTCTGTTGTCGATGACGCTTTCTG 3480A E V A R K Q L F K E L L A K V D G V VCGCAGAAGTTGCCCGCAAGCAGCTGT'TCAAGGAAC~TGCCAAGGTTGACGGCGTTGT 3D V R G R G L M L G V V L E R D V A K QAGACGTCCGTGGCAGGGGCTTGmGCGTGGTGCTGGAGCGCGACGTCGCAAAGCA 3600

A V L D G F K H G V I L N A P A D N I IAGCTGTTCTTGATGGTT'TTAAGCACGGCGTTATITIWATGCACCGGCGGACMCATTAT 3660R L T P P L V I T D E E I A D A V K A ICCGTTTGACCCCGCCGCTGGTGATCACCGACGAAGAAATCGCAGACGCAGWGGCTAT 3720argFA E T I A * M T S Q P Q V R H

TGCCGAGACAATCGCATWCTCAAACTTATGACTTCACAACCACAGGTTCGCCATT 3780F L A D D D L T P A E Q A E V L T L A AT T C T G G C T G A T G A T G A T C T C C - A G C A G G C A G A G G ~ A C C C T A ~ C G C A A 3840K L K A A P F S E R P L E G P K S V A VAGCTCAAGGCAGCGCCGTTTTCGGAGCGTCCA-ACCAAAGTCCGmAGTTC 3900L F D K T S T R T R F S F D A G I A H LTTTTTGATAAGACTTCAACTCGTACWGCTTCTCCT'TCGACGCGGGCATCGCTCAmG 3960G G H A I V V D S G S S Q M G K G E S LGTGGACACGCCATCGTCGTGGATTCCGGTAGCTCACAGATGGGTAAGGGCGAGKC- 4020Q D T A A V L S R Y V E A I V W R T Y A~ACACCGCAGCTGTATTGTCCCGCTACGTGGAAGCAATTGTG~CGCACCTACGCAC 080H S N F H A M A E T S T V P L V N S L SACAGCAA'MTCCACGCCATGGCGGAGACGTCCACTGTGCCGCTGGTGAACTCCTTGTCCG 140D D L H P C Q I L A D L Q T I V E N L SATGATCTGCACCCATGCCAGATTCTGGCTGATCTGCAGACTATCGTGGWCCXAGCC 4200P E E G P A G L K G K K A V Y L G D G DCTGAAGAAGGCCCAGCAGGCCTTAAGGGTAAGAAGGCTGTGTACCmGAmGACA 4260N N M A N S Y M I G F A T A G M D I S IACAACATGGCCAACTCCTACATGA'PTGGCTTXCCACCGCGGGCATGGATAmCCATA 4320I A P E G F Q P R A E FTCGCTCCTGAAGGGTTCCAGCCTCGTGCGGAUE 4355

. RBSRd

xhol

M

FmRl

Fig. 3. For legend see facing page.

104


7/10

Arginine biosynthesis in Corynebacterizlm glzltamictlm

C.gl.B.st.B.sub.N.g.




c.g1.B.st.B.sub.N.g.





MA------------EKGITAPKGFVASATTAGIKASGNPDMALWQGPEMTITKQTGQVTAVADGlVVPEGFQAAGWGLRYS-KNDLGVILCDVPAM-IQLSEDQIVKVT-GDVSSPKGFQAKGVHCGLRYS-KKDLGVIISETPAMAVNLTEKTAEQLPDIDGIALYTAQAGVKKPG-----HTDLTLIAVAAGS* * * . . .FSAAAVFTRNRVFAAPVKVSRENVA?XQ-IRAVLYNAGNANACNGLWm-VSAAVYTQSHFQAAPIK"QDSLKHGPTLKAVIWSAIANACTGEQGLK- T V G A V F T T N R F C A A P V A I A S H L F D E D G V R A L V I " A A Q G R I-SAAAWTQSHFQAAPLKVTQASLAVEQKLQAVIVNRPCANACTGAQGLK

, , * . - * * * * * *. * * . * . . * * * . . . . .DARESVSHLAQNLGLEDSDIGVCSTGLIGEIILPMDKLNAGIDQLTAEAPLD A Y T M R E S F A S Q L G I E P E L V A V S S T G V I G E H L D M E K I H A G K E - - T PDALAVCAAAARQIGCKPNQVNPFSTGVILEPLPADKIIAALPKMQPAFWNDAYEMRELCAKQFGLALHHVAVASTGVIGEYLPMEKIRAGIKQLVPGVTM

* * ' . * . * * * . * * * . * . 7 . . .GDNGAAAAKAIM'ITDTVDKET---VVFADGW!FV--GGMGKGV~PSLAADAEAFQ-TAILTTDTVMKRACYQTI'IDGK-TVTVGGMGSGMIHPNMAAGSGDFE-EAILTTDTVIKQTCYELAIGGK-TVTIGGARKGSWIHPNMAEAA-----RAIMTl'DTVPKAASREGKVGDQHTVRATGIAKGSGM* * . * * * * * . ** * * * * . * . . *TMLVCLTTDASVTQEMAQIALANATAVTFDTLDIDGSTSTNDTVFLLASGTMLMITTDANVSSPVLHAALRSITDVSFNQITVDGDTSTNDMVVVMASGTMLGFVTTDAAIEEKALQKALRETTDVSFNQITVDGETSTNDMVLWATMLGFIATDAKVSQPVLQLMTQEIADETFNTITVDGDTSTNDSFVIIATG* ** . . * * * . , * . , , * * * * * * * . . * .ASGIT-------PTQDELNDAVYAACSDIAAKLQADAEGVTKRVAVTWGLAGNDELTP-DHPDWENFYEALRKTCEDLAKQIAKDGEGATKLIEVRVRGCAENECLTE-DHPDWPVFKKALLLTCEDLAKEIARDGEGATKLIEAQVQGKNSQSEIDMIADPRYAQLKELLCSLALELAQAIVRDGEGATKFITVRVEN. . * . * . * * * * . ** . .TTNNEQAINAARTVARDNLFKCAMFGSDPNWGRVLAAVGM-ADMEPEKAKTDEEAKKIAKQIVGSNLVKTAVYGADANWGRIIGAIGYSD-AEVNPDNAKNNLDANVIAXKIVGSNLVKTAVYGTDANWGRIIGAIGHSA-AQWAEEAKTCDEARQAAYAAARSPLVKTAFFASDPNLGKRLAAIGYADVADLmDL* * * . . . * . * * . . . * . * . * . . .*. .ISVFFNGQAVCLDSTGAPGAREVD----LSGADIDVRIDLGTSGEGQATVVDVAIGPMVMLK-GSEPQPFSEEFAAAYLQQETWIEVDL-HIGDGVGVAVEWLGGQCLFK-NNEPQPFSESIAKEYLEGDEITIVIKM-AmDGNGRAVEmLDDILVAEHGGRAASYTEAQGQAVMSKDEITVRIKL-HRGQAAATV_* . . . . . * .. . . .R"DLSFSWE1NSAYSS 388WGCDLTYDYVKINASYRT 4 0WGCDLTYDYIKINASYRT 40 6YTCDLSHGYVSINADYRS 406* * * * . * * , * .

38494745

87989694

13 714814414

182196192189

2324 624 223

27295291289

32434 434 033 9

37 039 238 838 8

Fig. 4. Comparison of entire amino acid sequences of ornithineacetykransferases by CLUSTAL v alignment. C.gl., C. g/utamicum(this work); B.st., B. stearothermophilus (Sakanyan et a/. ,1993a); Bsub., B. subtilis (O'Reilly & Devine, 1994); N.g., N.gonorrhoeae (Martin & Mulks, 1992). Asterisks indicate thatidentical residues occur in al l four polypeptides; dots showreplacement by similar amino acids.

arg/ and the argB genes (Fig. 2 ) . Nevertheless, no E. co l iK12 XSlD2R(pEX4) transformants could be selected onarginineless synthetic medium supplemented withampicillin. Arginine prototrophic E . coli K12XB25(pEX4) transformants, however, were readilyobtained.The orientation of the C. glzltamiczlm arg/ gene, inopposition to the lac promoter-directed transcription inpEX4, is probably responsible for the contrasting argEcomplementation results obtained with pPP2 and pEX4.The ornithine acetyltransferase activity displayed by pPP2harbouring E . co l i strains would then result from somepromoter located in the pBR327 vector itself.

Transcription of the C. gltltamictlm argB gene in E. coli ,however, seems independent from an extraneous pro-moter, a result confirmed by the significant N-acetylglutamate kinase activity observed in the E . coliK12 XB25(pGAB4) strain (Table 2). The pGAB4 plasmidwas constructed by inserting the 1.9 kb HindIII-XboIfragment (see Fig. 2) in HindIIIISalI double-digestedpromoter-probe vector pGA46. As no transcription canproceed from the pGA46 vector into the inserted DNA(An & Frisen, 1979), it seems likely that in E . coli cells atleast, transcription can be initiated at a promoter locatedbetween the Hind111 site at 1172 nt and the beginning ofthe structural argB gene of C.gltltamiczlm. Evidence for theoccurrence of a promoter site recognized by E. coli RNApolymerase preceding the argB structural gene has beenreported for B. stearotbermopbiltls as well (Sakanyan e t al.,1993b).

Repression of enzyme form ation and feedbackinhibition by arginineN-Acetylglutamate synthase, N-acetylglutamate kinaseand orn ithine acetyltransferase activities were measured inextracts of C.gltltamiczlm cells grown in the absence or inthe presence of arginine (Table 2). The levels of N-acetylglutamate synthase and ornithine acetyltransferasewere not affected by arginine addition. A fivefold re-pression of N-acetylglutamate kinase synthesis wasobserved in the presence of arginine.Inhibition of enzyme activity by arginine was tested forthe three enzymes by adding L-arginine (concentrationrange 0.01-100 mM) to the reaction mixture of the enzymeassays described above. Arginine was found to inhibit N -acetylglutamate synthase and N-acetylglutamate kinaseactivities ; he arginine concentrations for 50 YO nhibitionwere 40 and 2 mM, respectively. Feedback inhibition byarginine of corynebacterial N-acetylglutamate kinase hasalready been reported (Udaka, 1966). Inhibition by L-arginine (more than 80YOat 10 mM arginine) could alsobe detected for C. gltltamictlm N-acetylglutamate kinasesynthesized in E. coli K12 argB mutant cells carryingpPP2, pKS-1.9 or pGAB4. Whether N-acetylglutamatekinase inhibition actually plays a regulatory role in vivoremains to be established, since the apparent inhibitionconstant of arginine is high. One must, however, take intoaccount that this value was determined with crude extractsand at saturating levels of substrates.Inhibition by ornithine was tested for the enzymes listedin Table 2, except acetylornithinase. No inhibition couldbe detected for the first or the second biosyntheticenzymes, either in C.gltltamictlmor in recombinant E. colicell extracts. The corynebacterial ornithine acetyl-transferase activity was inhibited by ornithine (productinhibition); the apparent Ki value at saturating levels ofsubstrates was 5 mM.

Fig. 3. Nucleotide sequence of the 4.4 kb DNA region of C. glutamicum ATCC 13032 with the predicted amino acidsequences encoded by the argCJBDFgenes cluster. The potential ribosome-binding site (RBS) and selected restriction sitesreferred to in Fig. 2 are underlined. Stop codons are marked with asterisks.105


8/10


Table 2. Specific activities of four arginine biosynthetic enzymes in C. glutamicum and in E. coli strains carrying C.glutamicum arg genesValues are the means of at least two measurements made on independent cultures. The values for replicate assays differed from the meanby < 20 % for N-acetylglutamate synthase and ornithine acetyltransferase, an d by < 10YO or the other activities.

Strain Arginineaddition*

Specific activity [units (mg protein)-']

N-Acetylglutamate N-Acetylglutamate Ornithine Acetyl-synthase kinase acetyltransferase ornithinaseC.glutamicumATCC 13032

E . coli K12XA 4X A4(pPP2)XB25XB25(pPP2)XB25(pPP2)XB25(pKS-1.9)XB25(pGAB4)XB25[pBluescript I1 KS ( +)]X Sl D 2RXSlD2R(pPP2)

XB25(pKS-1*9)

0.060.06

< 0.001< 0.001N DN DN DN DN DN DN DN DN D

0.100.02N DN D< 0.0052.12.12.81.32.5< 0.005N DN D

0.160.12

< 0.0010.36N DN DN DN DN DN DN DN DN D

N D

N D

N DN DN DN DN DN DN DN DN D< 0.001< 0.001

ND , Not determined.* Arginine was added to synthetic me dium at a concentration of 5 mM. Succinate (0.5 YO ,W / V )was added for E. coli XSlD2R strains. E. colistrains carrying plasmids were grow n in the presence of am picillin.

Evolutionary relationships between enzymes of th eacetyl cycle could be detected between the acetylglutamate synthase ofN. crassa (Y. Yu & R. L. Weiss; EMBL accession numberComparison based on the Pearson & Lipman algorithm(1988) fails to reveal any significant similarity between thesequences of C.glzltamiczlm ornithine acetyltransferase andknown N-acetylglutamate synthases. This result enforcesour earlier suspicion that in spite of their functionalrelatedness the argcl and arg/ gene products belong todifferent evolutionary families (Sakanyan e t al., 1993a).FASTA comparisons (Pearson & Lipman, 1988) with theregistered polypeptide sequences do not point to any clearaffiliation of the ornithine acetyltransferases. On the otherhand similarities between the N-terminal part of ArgAand the ArgB polypeptide sequence have recently beennoticed (Reith & Munholland, 1993; Gessert e t al., 1994).These similarities appear in fact most pronounced whenE . co l i ArgA and C.glatamiczsm ArgB amino acid sequencesare compared: application of the Pearson & Lipman(1988) RDF2 program establishes a similarity value that is15 standard deviations above the mean value obtainedwith 100 random permutations of either sequence, ahighly significant value. It may therefore be assumed thatacetylglutamate synthases and kinases are indeedevolutionarily related.An alignment of the known ArgA and ArgB polypeptidesequences (data not shown) indeed reveals several highlyconserved amino acids. Surprisingly, no direct relatedness

L35484) and those of other organisms : he N. crassa ArgAenzyme seems weakly similar to kinase sequences only.The other known acetylglutamate synthase polypeptidesequences are obviously longer than the kinases by astretch of about 100-140 amino acid residues, and somehighly conserved regions occur in this part. The results ofa FASTA search of the registered sequences in the Swiss-Prot database suggests an intriguing affiliation. Indeed, ahigh similarity between this region of the E . coli acetyl-glutamate synthase and a 153 amino acid ORF, foundnext to the trpGDC cluster in Axospirillnm (Zimmer e t al.,1991), which in its turn can be related to the E. coli Rim1enzyme that acetylates the N-terminus of the ribosomalS18 gene (Yoshikawa e t al., 1987) and the Streptomjceslavenddae StaT protein, an acetylCoA-dependent acetyl-transferase (Horinouchi e t al., 1987). The A.pspirillzmzORF is, moreover, highly similar to the E. coli PhnOprotein (Makimo e t a!., 1991), which is involved inalkylphosphonate utilization that again appears related tovarious other acetyltransferases. These similitudes mightreflect an ancient link between the argcl-encoded enzymeC-terminal part and a family of small acetylCoA-dependent acetyltransferases.New insights into the function and the evolution of theacetyl cycle are presently being looked for in a com-parative analysis of structure-function relationships

106


9/10

A r g i n i n e b i o s y n th e s i s i n Curynebacterizrmgltltamiczrm

in t h e m o n o f u n c t i o n a l ornithine ace t y l t r ans f e r a se s ofC.gltltamictlm disclosed in this work and the bifunctional,h o m o l o g o u s enzyme of B. stearuthermuphiltls ( S a k a n y a n e tal., 1993a ) .

ACKNOWLEDGEMENTSWe are indebted to D. Gigot for the synthes i s of ol igo-deoxyr ibonucleotides . This w ork was par t ia l ly su ppor ted by aGrant f rom the Rtgion des Pays de la Loi re (Cont ra t de PlanEtat-Rkgion) , by the Belgian Found at ion for Fund amenta l andJoint Research (FRFC-FKFO), and by an OZR-fund of theVrije Universiteit Brussel.

An, G. & Friesen, 1. D. (1979). Plasmid vehicles for direct cloning ofEscherichia coli promoters. J Bacteriol140, 400-407.Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extractionprocedure for screening recombinant DNA. Nucleic Acids Res 7 ,

Bolivar, F. (1978). Construction and characterization o f new cloningvehicles. 111. Deriv atives of plasmid pBR322carrying u nique EcoRIsites for selection of EcoRI generated recombinan t D N A molecules.Gene 4, 121-130.Boonchird, C., Messengey, F. & Dubois, 5. (1991). Characterizationof the yeast ARG5,6 gene: determination of the nucleotidesequence, analysis of the control region and o f ARG5 ,6 transcript.M o l 6 Gen Genet 226, 154-1 66.BrOer, S., Eggeling, L. & Krlmer, R. (1993). Strains ofCor_ynebacteriumglutamicum with different lysine productivities mayhave different lysine excretion systems. A p p l Environ Microbiol59,Brown, K., Finch, P. W., Hickson, 1. D. & Emmerson, P. T. (1987).Complete nucleotide sequence of the Escherichia coli argA gene.Nucleic Acids Res 15, 10586.Cunin, R., Glansdorff, N., Pierard, A. & Stalon, V. (1986).Biosynthesis and metabolism of arginine in bacteria. Microbiol RevDavis, R. H. (1986). Comp artmental and regulatory m echanisms inthe arginine pathways of Neurospora crassa an d Saccharomycescerevisiae. Microbiol Rev 50, 280-313.Dharmsthiti, S. & Krishnapillai, V. (1993). DNA sequence con-servation at the gen e level in a conserved chrom osoma l segment intw o Pseudomonas species. J Genet 72 , 1-14.Eikmanns, B. J., Kircher, M., Liebl, W. & Reinscheid, D. J. (1991).Discrimination of Coynebacterium glutamicum, Brevibacterium flavuman d Brevibacterium lactofermentum by restriction pattern analysis ofDNA adjacent to the hom gene. FE M S Microbial Lett 82 , 203-208.Gessert, 5. F., Kim, J. H., Nargang, F. E. & Weiss, R. L. (1994). Apolyprotein precursor of t wo mitochondria1 enzymes in Neurosporacrassa. Gene structure and precursor processing. J Biol Chem 269,Haas, D. & Leisinger, T. (1974). Multiple control of N-acetylglutamate synthetase from Pseudomonas aeruginosa : synergisticinhibition by acetylglutamate and polyamines. Biochem Biophys ResCornmha 60,4247.\Haas, D. & Leisinger, T. (1975). N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regu-latory properties. Eu r J Biochem 52, 377-383.Haas, D., Kurer, V. & Leisinger, T. (1972). N-acetylglutamate

1513-1 523.

31 6-321.

50, 314-352.

81 89-8203.

synthetase of Psetldomonas aeruginosa. An assay in vitro and feedbackinhibition by arginine. Eu r J Biochem 31, 290-295.Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL : a package forperforming multiple sequence alignment on a microcomputer. GeneHindle, Z., Callis, R., Dowden, S., Rudd, B. A. M. & Baumberg, S.(1994). Cloning and expression in Escherichia coli of a Streptomycescoelicolor A3(2) argCJB gene cluster. Microbiology 140, 31 1-320.Hoare, D. 5. & Hoare, 5. L. (1966). Feedback regulation of argininebiosynthesis in blue-green algae and photosynthetic bacteria. JBacteriol92, 375-379.Horinouchi, S., Furuya, K., Nishiyama, M., Suzuki, H. & Beppu, T.(1987). Nucleotide sequence of the streptothrycin acetyltransferasegene from Streptomyces avendulae and its expression in heterolo goushosts. J Bacterioll69, 1929-1 937.Jones, D. & Collins, M. D. (1986). Irregular, nonsporing Gram-positive rods. In Bergty's Manual o f Systema tic Bacteriology,vol. 2, pp .1261-1434. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe &J . G. Holt. Baltimore: Williams & WilkinsKinoshita, 5. & Nakayama, K. (1978). Amino acids. In PrimayProducts of Metabolism, p. 209-261. Edited by A. H . Rose. London,New York & San Francisco: Academic Press.Makino, K., Kim, S., Shinagawa, H., Amemura, M. & Nataka, A.(1991). Molecular analysis of the cryp tic and functionalph n operonsfor phosphonate use in Escherichia coli K-12. J Bacteriol 173,Malumbres, M., Gil, 1. A. & Martin, 1. F. (1993). Codon preferencein Corynebacteria. Gene 134, 15-24.Martin, P. R. & Mulks, M . H. (1992). Sequence analysis andcomplementation studies of the argJ gene encoding ornithineacetyltransferase from Neisseria gonorrhoeae. J Bacteriol 174,2694-2701.Meile, L. & Leisinger, T. (1984). Enzym es of arginine biosynthesisin methanogenic bacteria. Experientia 40, 899-900.Miller, J. H. (1972). Experiments in Molecular Genetics. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory.Moun tain, A., M ann, N. H., Munt on, R. N. & Baumberg, S.(1984).Cloning of a Bacillus subtilis restrict ion fragment complementingauxotrophic mutants of eight Escherichia coli genes of argininebiosynthesis. M o l 6 Gen Genet 197, 82-89.Mou ntain , A., McChesney, J., Smith, M. C. M. & Baumberg, 5.(1 986). Gene sequence encoding early enzymes of arginine synthesiswithin a cluster in Bacillus subtilis, as revealed by cloning inEscherichia coli.J Bacterioll65, 1026-1 028.O'Reilly, M. & Devine, K. M. (1994). Sequence and analysis of thecitrulline biosynthetic operon argC-F from Bacillus subtilis. Micro-Parsot, C., Boyen, A., Cohen, G. N. & Glansdorff, N. (1988).Nucleotide sequence of Escherichia coli argB an d argC genes:comp arison of N-acetylg lutamate kinase and N-acetylglutamate-y-semialdehyde dehydrogenase with homologous and analogousenzymes. Gene 68, 275-283.Pearson, W. R. & Lipman, D. 1. (1988). Improved tools forbiological sequence comparison. Proc Natl Acad Sci U S A 85,2444-2448.Picard, F. J. & Dillon, J. R. (1989). Cloning and organization ofseven arginine biosynthesis genes from Neisseria gonorrhoeae. JBacterioll71, 1644-1651.Reith, M. & Munholland, J. (1993). Two amino-acid biosyntheticgenes are encoded on the plasmid gen ome of the red alga Porphyraumbilicalis. Curr Genet 23, 59-65.

73 , 237-244.

2665-2672.

biolog),140, 1023-1025.

107


10/10

V. S A K A N Y A N a n d OTHERS

Sakanyan, V. A., Hovsepya n, A. S Mett, 1. L. Kochikyan, A. V. &Petrosyan, P. K. (1990). Molecular cloning and structural-func-tional analysis of the arg inine biosynthesis genes o f the therm ophilicbacterium Baciflus stearotbermopbifus. Genetika 26 , 1915-1 925.Sakanyan, V., Kochikya n, A., M et t, I., Legrain, C. Charlier, D.,Pierard, A. & Glansdorff, N. (1992). A re-examination of thepathway for ornithine biosynthesis in a thermophilic and twomesophilic Baciffusspecies. J Gen Microbiof 138, 125-130.Sakanyan, V., C harlie r, D., L egrain, C., Koch ikyan, A., M ett , I.,PiBrard, A. & Glansdorff, N. (1993a). Primary structure, partialpurification and regulation of key enzymes of the acetyl cycle ofarginine biosynthesis in Bacillus stearotbermopbilus dual function ofornithine acetyltransferase. J Gen Microbiof 139, 393-402.Sakanyan,V. A,, Legrain, C Charlier, D., Kochik yan , A. V., Osina,N. K. & Glansdorff, N. (1993b). N-acetylglutamate-5-phospho-transferase of the thermo philic bacterium Baciflusstearotbermophihs:nucleotide sequence and enzyme characterization. Genetika 29,Sakanyan, V., Desmarez, L. Legrain, C Cha rlier, D., M et t, I.,Koch ikyan , A. Savchenko,A., Boyen, A., Falm agne, P. Pierard, A.& Glansdorff, N. (1993~). Gene cloning, sequence analysis,purification, and characterization of a therm ostable aminoacylasefrom Bacillus stearotbermopbifus. A pp f Environ Microbiof 59,Sambrook, J Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning :a Laborat07 Manual, 2nd edn. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory.Sanger, F Nicklen, 5. & Coulson, A. R. (1977). DNA sequencingwith chain-terminating inhibitors. Proc Natl Acad Sci U S A 74 ,Shiio, I., Otsuka, 5.1. & Takahashi, M. (1962). Effect of biotin onthe bacterial formation of glutamic acid. I. Glutamate formationand cellular permeability of amino acids. J Biocbem 51, 56-62.Shinners, E. N. & Catlin, B. W. (1978). Arginine biosynthesis inNeisseriagonorrboeae : enzymes catalyzing the formation of ornithineand citrulline. J Bacterioll36, 131-135.Udaka, 5. (1966). Pathway-specific pattern of control of argininebiosynthesis in bacteria. J Bacteriof 91 , 617-621.

556-570.

3878-3888.

5463-5467.

Udaka, S. & Kinoshita S. (1958). Studies on L-ornithine fer-mentation. I. Th e biosynthe tic pathway of L-ornithine in Micrococcusgfzltamicus.J Gen Appf Microbiof 4, 272-282.Van de Casteele, M., Dem arez, M., Legra in, C Glansdorff, N. &Pierard, A. (1990). Pathways of arginine biosynthesis in extremethermophilic archaeo- and eubacteria. J Gen Microbiof 136,Van Huffel, C. Dubois, E. & Messenguy, F. (1992). Cloning andsequencing of arg3 and argl 1 genes of Scbixosaccbaromycespombe o na 10-kb DNA fragment. Heterologous expression and mito-chondrial targeting of their translation products. Eu r J BiocbemVieira, 1. & Messing, J. (1982). The pUC plasmids, a M13mp7-derived system for insertion mutagenesis and sequencing withsynthetic universal prim ers. Gene 19, 259-268.Vogel, H. 1. & MacLellan, W. L. (1970). Acetylornithinase ( E . coli].Methods Enzgmof 1 7A , 265-269.Wipf, B. & Leisinger, T. (1979). Regulation of activity and synthesisof N-acetylglutamate synthase from Saccbaromyces cerevisiae. JBacteriof 140, 874-880.Yamada, K. & Komagata K. (1970). Taxonomic studies oncoryneform bacteria. 111. DNA base composit ion of coryneformbacteria. J Gen A p p f Microbiof 16, 55-65.Yanisch-Perron, C. Vieira, 1. & Messing, J. (1985). Improved M13phage cloning vectors and host strains : nucleotide sequences of theM13mp18 and pUC19 vectors. Gene 33, 103-119.Yos hika wa A., Isono, S., Sheback, A. & Isono, K. (1987). Cloningand nu cleotide sequencing of the genes rimI an d rim] which encodeenzymes acetylating ribosomal proteins S18 and S5 of Escbericbiaco f i . Mof & Gen Genet 209, 481-488.Zimm er, W., A paricio, C. & Elmerich, C. (1991). Relationshipbetween tryptophan biosynthesis and indole-3-acetic acid pro-duction in Axospirilfum dentification and sequ encing of a trpGDCcluster. Mof & Gen Genet 229, 41-51.

1177-1 183.

205, 33-43.

Received 1 August 1995; accepted 25 August 1995.

108

Genes and Enzymes of the Acetyl Cycle of Arginine Bio Synthesis in C. cum Enzyme Evolution in the...

Documents

Transcript of Genes and Enzymes of the Acetyl Cycle of Arginine Bio Synthesis in C. cum Enzyme Evolution in the...