Gene. 90 (1990) 31-41 Elsevier

31

GENE 03534

C l o n i n g a n d c h a r a c t e r i z a t i o n o f the histidine biosynthetic g e n e cluster o f S t r e p t o m y c e s coelicolor A3(2) (Recombinant DNA; Escherichia coil; operons; primary metabolism; protein homology)

Danila Limauro', Alessandra Avitabile', Carmela Cappellano b, Anna Maria Puglia b and Carmdo B. Brnni" ° Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R.. Dipam'mento di Biologia e Patologia Ceilulare e Molecolare. Univers~ a~" Napoli. 80131 Naples (Italy), and b Dipartimento di Biologia Cellulare e delio Sviluppo, Universit~ di Palermo, 90123 Palermo (Italy) Tel. (91)6161201 Received by K.F. Chater: 27 October 1989 Accepted: 19 February 1990

SUMMARY

Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coil hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment ofgenomic DNA was further characterized. When cloned in the plasmid vector, plJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coll. The order of the genes in S. coelicoior A3(2) (5'-hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coll.

INTRODUCTION

In S. coelicolor A3(2) the structural organization ofthe h/s genes is intermediate in its complexity between that of enterobacteria, where the eight genes are clustered in a single operon (Carlomagno et al., 1988), and that of lower eukaryotic cells, where each gene resides on a distinct chroCorrespondenceto: Dr. C.B. Bruni, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universit/t di Napoli, Via S. Pansini 5, 80131 Naples (Italy) Tel. +39(81)7462047; Fax +39(81)7703285. Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); cpm, counts/rain; d, distal region of gene; A, deletion; h/~, genes involved in histidine biosynthesis; kb, kilobase(s) or 1000bp; LB, Luria-Bertani (medium); MM, minimal medium; nt, nucleotide(s); ORF, open reading frame; p, proximal region of gene; R, resistant; S., Streptomyces; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/0.015 M Na3" citrate pH 7.6; Th, thiostrepton; wt, wild type; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990ElsevierSciencePublishersB,V.(BiomedicalDivision)

mosome (Broach, 1981). Previous genetic and biochemical studies indicated that in S. coelicolor A3(2) the his genes map at three different loci on the chromosome: a 'cluster' of 5/6 genes map at the 12 o'clock position, one or two genes map at the 2 o'clock position and a single gene (h~D) maps close to the 6 o'clock position (Carere et al., 1973; Derkos-Sojak et al., 1985; Hopwood et al., 1985; Russi et al., 1973). Preliminary evidence that these genes are subject to some form of regulation has been provided (Carere et al., 1973; Derkos-Sojak et al., 1985; Russi et al., 1973), but the molecular details of this, and indeed of most of the pathways involved in primary metabolism of Streptomyces, are as yet unknown. Other more subtle differences in the structural organization of individual cistrons are also observed in several species. In the enterobacteria, three ofthe hLvcistrons (hisD, hisB and hisIE) code for bifunctional enzymes (Brenner and Ames, 1971; Chiariotti et al., 1986a), whereas in lower

32

eukaryotes the hisB activities reside on separate genes (Broach, 1981) while the hisD and hislE activities remain encoded by a single gone (Donahue et al., 1982; Legerton and Yanofsky, 1986). In contrast, in archaebacteria the hislE activities are encoded by separate genes (Beckler and Reeve, 1986). In Bacillus subtilis all the his genes, with the exception of hisH, are clustered in a small region of the chromosome (Piggot and Hoch, 1985). In Streptomyces the hisB activities and possibly the hisIE activities are encoded on separate genes (Hopwood et al., 1985). "I~e reasons and evolutionary significance, if any, for these different structural organizations are as yet unknown. We have begun to investigate the structure and expression of the his genes in S. coelicolor A3(2). The aim of the present work was to clone the genomic region spanning the major his cluster, to study the complementation of auxotrophs ofE. coli and ofS. coelicolor A3(2), and to sequence a 4-kb his DNA fragment.

RESULTS AND DISCUSSION

(a) Cloning of the Streptomyces coelicolor A3(2) hisB gene The S. coelicolor hisB gene is part of the his cluster (Fig. 1); it encodes the seventh enzyme in the biosynthetic pathway (imidazole glycerolphosphate dehydratase; EC his GENES IAl.lcl~tal,I

E. coil

$. coellcolor AS (2)

Fig. !, Organization of the his structural genes on the E, coil and S, coeli. color A3(2) genetic maps. The genes are clustered in a single operon in E. coil at map position 44.1 min (let~) (Bachmann, 1987). The his genes ofS. coelicolor A3(2) have been mapped in at least three different regions

of the chromosome (dght) (Hopwood et al., 1985). The nomenclature of the His genetic loci in the two species is different and, therefore, rather confusing. In fact: £. coil hisG was tentatively named h~H in S. coelicolor (Russi et al., 1973) and has disappeared from more recent maps (Hopwood et al,, 1985); E. coil genes (first) correspond to the following S. coelicolor genes: hisD = hisA; hisC -- hisG; hisB (bifunctional hisBpx and hisBd) ~- hisD and hisB; hisH = hisC or hisF or hisl; hisA = hisC or hisF or h/.vl; hisF = hisC or hisF or hisl; hislE (bifunctional) = hisE and nn-idantified gene.

4.2.1.19), and corresponds to the C-terminal domain (hisBd) of the single bifuncfional hisB gone of enterobacteria (Chiariotti et al., 1986b). It has been shown that his structural genes from several, even distantly related, species can complement E. coil his auxotrophic strains (Bazzicalupo et al., 1987; Beckler and Reeve, 1986; Cue et al., 1985; Legerton and Yanofsky, 1986; Struhl et al., 1976). We isolated the S. coelicolor hisB gone from a library of chromosoma] DNA that had been partially digested with Sau3AI and cloned in the vector pUC12; hybrid plasmids capable of conferring Ap resistance and of complementing the hisBd mutation orE. coil strain FB251 (Grisofia et al., 1982)were selected. One plasmid, pSCH 1 (Table I), was isolated and further characterized. Digestion of the plasmid DNA with EcoRI + HindIII generated two fragments of 2.7 (vector) and 3.4 (insert) kb. To verify the origin of the cloned insert and to characterize it further, we performed the following experiments. The 3.4-kb DNA fragment was purified, labeled by nick translation (Rigby et al., 1977) and used as a probe on Southern blots (Southern, 1975) of E. colt and S. coelicolor genomic DNA cleaved with BamHI. The probe did not hybridize to E. coil DNA, whereas strong hybridization to an S. coelicolor DNA fragment larger than 12 kb was observed (data not shown). The purified 3.4-kb EcoRI-HindIII fragment was cloned in opposite orientations in vectors pUCI8 and pUCI9. After transformation of strain FB251 with the resulting plasmids pSCHla and pSCHlb in rich medium, several individual Ap R clones were tested for growth on minimal plates. All the clones tested were prototrophic (Table II). These data indicate that the insert is a fragment of 5. coelicoior chromosomal DNA carrying the hisBd homologue, and that the gone is either expressed from an internal promoter or alternatively from plasmid promoters functioning in opposite orientations. Growth rate measurements did not help to solve this issue since strains carrying the pSCHla and pSCHIb recombinant plasmids grew with similar, rather long generation times (120 and 150 rain, respectively, compared to 60 min for the wt). Finally, the 3.4-kb DNA fragment was subcloned in the Streptomyces plasmid vector pLI699; the derivative plasmid pIJ64 was able to complement the hisB S. coelicolor auxotrophic strain 410. This same plasmid was able to complement strain 430 carrying a mutation in hisC, which is located in the h/s cluster, but could not complement strains 415 and 44 carrying mutations in hisG and hisA, respectively, which are also located in the cluster (Fig. 1; Tables I and II).

(b) Characterization of pSCHI and cloning of the Streptomyces coelicolor A3(2) his gene cluster To obtain further information on the structural organization of the S. coelicoior genomic region spanning the h/s gene cluster, we first established a detailed restriction map

33 TABLE I Vectors and plasmids carrying portions of the hb gene cluster of Streptomyces coelicolorA3(2) Plasmid a

Vector

Insert b

pUCI2

3.4-kb Sau3AI

pUCI 8

3.4-kb EcoRI-H/ndlIl

pUCI9 pUCI9 pUCI2 pUCI2

3.4-kb EcoRI-HindlIl 3.l-kb Kpnl 6.5-kb H/ndlll-EcoRl 12-kb Sacl

plJ2925

3.4-kb EcoRI-H/ndlll

plJ699 plJ2925 plJ699

3.4-kb Bg/II 6.5-kb HindlIl-EcoRI 6.5-kb Bg/ll

pUCI2 pSCH! pUCI8 pSCH ! a pUCI9 pSCH lb pSCH28 pSCH3328 pSCH31 plJ2925 plJ63 plJ699 pIJ64 pIJ72 pIJ73

Source or reference Messing and Vieira (1982) This work. Partial Sau3Al genomic fragment Yanisch-Perron et al. (1985) This work. From pSCHI Yanisch-Perron et al. (1985) This work. From pSCHI This work. Kpnl genomic fragment T h i s work © This work. SacI genomic fragment (MJ. Bibb) d T h i s work. From pSCH! Kieser and Melton (1988) This work. From plJ63 This work. From pSCH3328 This work. From plJ72

a Streptomyces plasmids were purified according to Kieser (1984). E. coli plasmids were purified according to Clewell (1972) and finally banded on a CsCI/ethidium bromide equilibrium density gradient. See Fig. 2 and Table II. b DNA fragments were isolated on and purified from 5% polyaerylamide slab gels by electroeintion as described elsewhere (Maniatis et al., 1982) or from 1% agarose gels using the Ganeclean (Bio-101) system. The fragments are shown in Fig. 2. The 3.l-kb Kpnl fragment ofpSCH28 was cloned into the single Kjynl site ofpSCH! and the proper orientation was determined by restriction analysis. d plJ2925 is a pUCI8 derivative containing a polylinker from plJ486 (Ward et al., 1986) with flanking Bglll sites that make it possible to subclone fragments in the Streptomyces vector plJ699 (Kieser and Melton, 1988). i 1 kb HK B

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Fig. 2. Physical and genetic maps and recombinant plasmids of the h/s gene duster of S. coelicolor A3(2). Upper part: restriction maps of the three overlapping fragments cloned in the recombinant plasmids pSCH28, pSCH! and pSCH31. Recombinant plasmid pSCHI contains a Sau3Al.partiai genomic 3.4-kb fragment cloned in pUCI2 (Messing and Vieira, 1982). pSCH28 contains a KpnI genomic 3.l-kb fragment cloned in pUCI9 (YanischPerron et al., 1985) and pSCH31 contains a Sacl genomic 12-kb fragment cloned in pUCI2. These three plasmids were isolated from genomic libraries orS. coelicolorA3(2) DNA, Restriction maps of the plasmid DNAs were established using restriction sites uniquely present in the vectors. Fine mapping of DNA fragments was performed by double digestions with several restriction enzymes. The following sites are shown: B, BamHI; E, EcoRI; H, HindIll; K, Kpn I; Nc, NcoI; P, PstI; S, SacI; X, Xba I; Xh, Xhol; B*, hybrid restriction sites BamHllSau3AI resulting from the cloning procedure. Genomic banks were constructed by inserting partial Sau3AI digests (3- to 6-kb fragments) into pUCI2 (BamHI site), or KpnI fragments into pUCI9 (Kpnl site), or Sacl fragments into pUCI2 (Sacl site). The Sau3AI library was used to identify clones complementing the E. coli hisBd mutant FB251. The Kpnl and SacI libraries were used to transform E. coil strain DH5~ ( F - endA l hsdRI7 r~ m~ supE44 thi-I ;t- recA 1 gyrA96 relA 1 ¢80dlacZ~MI5), a derivative of strain DH5 (Hanahan, 1985) and the libraries screened by the in situ hybridization technique (Maniatis et ai., 1982), using as probe a 430-bp Pstl-$all fragment from pSCHI labeled to a specific activity of I x 109 cpm]/tg by the random priming DNA-labeling technique (Feinberg and Vogelstein, 1983). A 690-bp SacI-KpnI fragment from pSCH31 labeled to the same specific activity was used as a probe to screen the Kpnl library. The filters (Schleicher & Schuell) were washed under stringent conditions (0.1 x SSC/0.1% SDS, 65°C). Clones which gave a positive signal on duplicate filters were isolated and the plasmid DNA was purified. Lower part: expanded restriction map oftbe sequenced fragment. Additional sites shown: A, A/ul; Hc, H/ncll; N, Nael; SI, SalI; Sm, Sinai; Sp, Sphl. The thick lines below the restriction map identify the two DNA fragments which were used as probes. The open bar below the restriction map shows the relative position of the his genes and of the two putative ORFs (ORFI and ORF2) identified in the sequence. Dashed regions identify intracistronic elements. The genetic symbols of the his cistrous follow the nomenclature adopted for the enterobacteria (Bachmann, 1987). In parentheses are indicated the genetic symbols so far used for S. coelicolorA3(2) (Hopwood ¢t al., 1985).

34 TABLE !1 Complementation of his anxotrophic strains of Esche~chta colt and Strepwmyces coellcolor by several plasmids Strains a

Relevant host genotype a

Plasmids b

Complementation e

hbBd-

pSCHl pSCH la pSCHlb pSCH31

+ + + +

415

hb6 -

44

hbA -

410

hisB -

430

h/sC-

plJ64 plJ73 pL164 pIJ73 plJ64 plJ73 pIJ64 plJ73

+ + + + + +

E. co~ FB251

S. coeiicolor

a Recipient stralns:E, coliFB251 (hisB855recAS6)(Grisoliaet al., 1982), S. coelicolor 415 ( hiz6 8), $. coelkolor 44 ( hisA i ), S. coeltcolor 410 (pheA ! hisB2 strA 1), S. coeltcolor 430 (hisC9 adeA !) (kindly provided by D.A. Hopwood). Cultures of E. coil strains were grown at 37°C. Rich media were LB and 2 × YT (Miller, 1972), SOB and SOC (Hanahan, 1983). Minimal media were VBC (Vogel and Bonnet, 1956) and M9 (Miller, 1972) supplemented with 0.5% (w/v) glucose. Media were solidified with i.2~o (w/v) ager (Difco) or 0.7% (soft eger). When required, Ap (50 #8/ml) and L-histidine(0,1 raM) were added to both solid and liquid media. Streptomyces strains were grown according to the procedures described by Hopwood et el. (198S), R2YE medium and MM were the solid media used; YEME was used as liquid medium. When required, Th and His were added as specified by Hopwood et ill. (1985), htsBd, distal part of gone hlsB. b See Table I for details of the construction of the different plasmids, £, colt strains were transformed according to Hanahan (1983). To trans. form 8treptomyces strains protoplasts were obtained and treated according to Hopwood et el. (1985), To overcome the restriction system of S. coeltcolor A3(2) (MacNetl, 1988) the recombinant plasmids were first introduced into protoplasts of 8. Itvtdans strain 66 (John Innes collection No. 1326). The £. coil anxotrophic strain FB251 was transformed with the different plasmids and transformants were isolated on LB + Ap plates. Individualtransformants were tested for growth on minimal e Ap plates. Protoplasts of S. livtdons and of 8. coeltcolor auxotrophic strains were transformed with the different plasmids selecting for Th resistance. Regenerated protoplasts were tested for growth in MM.

o f the 3.4-kb S a u 3 A I fragment (Fig. 2), and used different restriction fragments as radioactive probes for Southern (1975) blots o f S. coelicolor genomic D N A cleaved with several restriction enzymes. This information, which was in agreement with the restriction m a p d a t a o f p l a s m i d p S C H 1 (Fig. 2), w a s used to c o n s t r u c t other genomic libraries to isolate overlapping genomic clones spanning the h/s cluster. T w o r e c o m b i n a n t plasmids, containing a 3.1-kb K p n I fragm e n t ( p S C H 2 8 ) a n d a 12-kb S a c I fragment ( p S C H 3 1 ) ,

Fig. 3. The nt sequence of the $, coellcolor A3(2) his gone cluster and the • derived aa sequence. The different genes and the two putative ORFs (ORFI and ORF2) are indicated on the right margin. Stop codons are indicated by triple asterisks, The putative Shine-Daigarno consensus sequences and the start codons of each cistron are underlined. The 4-kb SocI.Sau3AI genomic fragment containing the his genes was sequenced by the dideoxynucleotide method (Sanger et el., 1977). The entire fragment, cloned in plasmid pSCHI (nt 605-3981), and the Sacl-Kpnl fragment, derived from plasmid pSCH28 (nt 1-696), were used in the sequencing procedures. JM101 (supE tki ~ac-proAB[F'traD36 proAB + laclqZ .4M15]) was used as host for subcloning restriction fragments in bacteriophage M13mpi8 and Ml3mpl9 (Yanisch-Perron et el, 1985) vectors. Most of the sequence was determined on purified restriction fragments whose location was known from the restriction map. Gaps in the sequence were filled by shotgun sequencing of an Alul digest or by the use as primers ofad hoc constructed synthetic oligodcoxyribonucleo. tides. To overcome compression problems c!ueto the high G + C content, each sequence was determined more than once using different commercially available sequencing kits: Sequenase and TAQuence (United States Biochemical Co.) and T7 Sequencing Kit (Pharmacia LKB Biotechnology AB). The sequence was determined on both strands and all the restriction sites used to sub¢lone in the MI3 vectors were overlapped. Assembly and editing of the data, analysis of the completed sequence for ORFs, codon usage, restriction sites and homology comparisons with the EMBL GenBank, were performed using the Microgenie (Beckman) program and an IBM AT computer. The sequence has been deposited with GenBank and assigned the accession number M31628. For further discussion of the features of the sequence, see section e and Fig. !.

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T6AA600C6~C~TL~T0o~P~6~BU~CGC0o~CET~GC0o~0oi~CG~TT~|;TU1¢6CT~H~`TTC9TCTM6~C00rL~C0o~ ICT~.'~'o6~TC~ luLysThtTIuOrg~prLeuVllAlaGluLeurgA1al ie61ylyrllilVlLThr 61uSlrOsp01aAsnPheVa161rd~htGlyr§PO~All~l)Se~fli~laTSrTr0Arg L 1450 1460 1470 1480 1690 1~0 1510 1580 1030 1540 I~0 1560 A~A~CCTC6ACC6E66C6?~C166T~T00C000oT00CT6C006?Ci~CC6CC6r~r~CTCC00~166~0o9CBAC9CG~1CC~CGACGCG6~ACST6AA6?C&q6M8B y st ieLlusprg61y'41lLluVaiOrospAsn61yValPro61yTrpLluAroVllThrOi161yThrPr o61uBlusnAsp911PhlLeuAspA|lVllllr|OluVllLyst.'-~

2410 2430 9430 3440 9438 2q60 2470 9490 34~0 asoo ~10 L~o OI~3?CC6CT2CBLT, P~6CCC lP,~P~6C6C000O6CC60CGTC61~IIC~CCS'T60C?0C60CM00CC6?~C~|~T0OT~6~ ltSnValllrge+erAIdluSr|OlaLeuOtar801961y011Asp¥a 161u11etflrOrgOspTytllspLys9taHetOsn0h~pBlyteuLeuVaIPro61909161y0111~h~18

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38

were isolated and further characterized by restriction mapping (Fig. 2, Table I) and complementation assays (Table H). As expected, pSCH31 could complement E. coil auxotrophic strain FB251. A recombinant plasmid (pSCH3328) comprising the 3.1-kb KpnI fragment of pSCH28 and the 3.3-kb region ofplasmid pSCH 1, with the two fragments in their native configuration, was constructed. The resulting 6.5-kb insert was subcloned in Streptomyces plasmid vector pL1699 and the derivative plasmid pIJ73 was used in complememation assays. This plasmid was able to complement not only hisB and hisC, but also hisG and hisA S. coelicolor auxotrophic strains (Table II). These data demonstrated that the cloned region spanned at least part of the h/s cluster and gave preliminary information on the gene order (hisG, hisA) and (hisB, hisC). (c) Sequence of the Streptomyces coelicolor A3(2) his cluster and comparison with the his eistrous of Escberichia coil K-12 The nt sequence of a 3981-bp region from the left-hand SacI site of plasmid pSCH31 to the end of the cloned fragment in pSCH 1 was determined (Fig. 3). The G + C content of this region is 71% and is comparable to the average G + C composition of Streptomyces DNA (Gladek and Zakrzewska, 1984). The sequence of both strands was searched for ORFs using the Microgenie (Beckman) program. ORFs derived from the lower strard did not show significant homology with the gene products of the h/s operon of E. coll. On the contrary, of the seven contiguous ORFs encoded by the upper strand (Fig. 3), five did exhibit significant homologies ranging from 35-46% of aa sequence identity. When conservative aa substitutions are allowed (Dayhoff et ai., 1978)the similarity is higher, ranging from 55-62%. The deduced aa sequence of the first ORF at the 5' end is truncated at the N terminus and is 155 aa long. This sequence shows significant homology with the C-terminal region of the hisD gene product of E. coil, the second structural gene of the operon encoding the bifunctional enzyme L-histidinol : NAD ÷ oxidoreductase (EC 1.1.1.23), which catalyzes the tenth and eleventh steps of the biosynthetic pathway converting histidinol to histidinal and this aldehyde to histidine (Bt~rger and GOrish, 1981). This ORF, when aligned with the distal E. coil hisD sequence (aa 283-434) gives an overall identity of 37.5~o (Fig. 4). The similarity is not distributed evenly throughout the aa sequence; instead, there are regions which exhibit much higher level of similarity. In particular the regions from aa 43-101 vs. 323-381 and 129-146 vs. 409-426 are over 50% identical. It has been inferred that this region, which is also highly conserved between E. coil and the corresponding Saccharomyces cerevisiae enzyme (Bruni et al., 1986), corresponds to the domain responsible for the sec-

end enzymatic function, conversion of histidinal to histidine. These data, together with the complementation assays (Table II), indicate that the nt sequence encoding this ORF is the hisA gene of S. coelicolor A3(2), which corresponds to the hisD gene of the enterobacteria. The second identified 369-aa ORF shows significant homology with the hisC gene product of E. cell, the third structural gene of the operon encoding the enzyme histidinol-phosphate aminotransferase (EC 2.6.1.9), which catalyzes the eighth biosynthetic step of the pathway converting imidazolylacetolphosphate to L-histidinolphosphate (Winlder, 1987). This ORF, when aligned with the E. coil hLv-C-encoded 356 aa, gives an overall identity of 35?/o (Fig. 4). Also in this case regions of relatively higher similarity, probably involved in the catalytic function of the enzyme, are observed and are coincident with similar homologous regions found between the corresponding gene products orE. coil hisC and of S. cerevisiae HIS5 (Grisofia et al., 1986; Nishiwaki et al., 1987 and data not shown). These data, and the complementation assays (Table II), establish that this ORF is the S. coelicolor counterpart, previously named hisG (Russi et al., 1973), of the enteroo bacteria hisC gene. The start codon of this cistron appears to be GUG instead of AUG, although positive identification will require aa sequencing. In support of this assignment, aside from the alignment with the E. coli sequence, is the observation that in S. coelicolorthe G U G codon is used rather frequently as an initiator (Hopwood et al., 1986) and that the putative start codon is preceded (nt 456-459, Fig. 3) by consensus sequences for a ribosomal binding site (Shine and Dalgnrno, 1974; Bibb and Cohen, 1982). Another interesting feature is that the start codon overlaps with the stop codon of the preceding hisD cistron (GUGA). This compact organization of different cistrons is rather common for bacterial genes clustered in operons (Kozak, 1983). In particular, in the h/s operons of E. coil and S. typhimurium, with the exception of an intercistronic region between the first (hisG) and second (hisD) structural genes, all the remaining cistrons have overlapping translational termination and initiation signals (Carlomagno et al., 1988) indicative of translational coupling. The hisB gene of E. coil, the fourth structural gene of the operon, encodes a bifunctional enzyme. The promoter proximal region (hisBpx) encodes the histidinol phosphatase (EC 3.1.3.15) activity (N-terminal domain: aa 1-164) and the distal region (hisBd) encodes the imidazole glycerolphosphate dehydratase (EC 4.2.1.19) activity (C-terminal domain: aa 165-355). This enzyme catalyzes the seventh and ninth biosynthetic steps of the pathway converting imidazole giycerolphospT: ate to ha~dazolylacetolphosphate and L-histidinol phosph~te to L-histidinol (Chiariotti et al., 1986b). In other spec'es, such as S. cerevisiae (Broach, 1981) and S. coelicolor (Hopwood et al., 1985), the two

39

activities are encoded by separate genes. The cloned DNA fragments described here were able to complement both E. coli and S. coelicolor hisB dehydratase mutations (Table II). The third identified 197-aa ORF ofS. codicolor shows significant homology with the hisBd product. When this ORF was aligned with the C-terminal E. coli hisBd 190-aa sequence, an overall identity of 46.2~o was found (Fig. 4). Also in this case regions of higher similarity are present and these same domains are conserved in the enzyme encoded by the homologous HIS3 gene of $. cerevisiae (Stmhl, 1985; and data not shown). The AUG start codon is preceded by a canonical Shine-Dalgarno box (nt 1557-1562, Fig. 3) and overlaps with the stop codon of the preceding hisC gene (AUGA). The hisBd cistron is separated from the next his cistron by a DNA region of 209 nt (2165-2373 of Fig. 3). Within this region there is a putative ORF of 54 aa (ORFI, Fig. 3), which did not show any significant homology with other proteins in the Protein Data Bank or with his gene products of enterobacteria. The AUG start codon is separated by only two nt from the stop codon of~he preceding hisBdgene and is preceded by a canonical Shine-Dalgarno consensus sequence (nt 2149-2153). The derived aa sequence is in very good agreement with the rules governing codon usage in Streptomyces (Hopwood et al., 1986). The next identified 222-aa ORF shows significant homology with the hisH gene product of E. coil, the fifth structural gene of the operon encoding the enzyme glutamine amidotransferase. This ORF, when aligned with the E. coil hisH.encoded 196 aa, gives an overall identity of 42.5 ~ (Fig. 4). Also, regions of relatively higher similarity, probably involved in the catalytic function of the enzyme, •are observed. It was not possible to determine unambiguously the site oftranslational initiation. The one shown, UUG, overlaps with the stop codon of the preceding ORF 1 (UUGA) and is preceded by a canonical Shine-Dalgarno box (nt 2217-2221, Fig. 3). U U G is used as a start codon at low frequency (1%) in E. coli (Gold and Stormo, 1987). The next identified 240-aa ORF starts 17 nt after the stop codon ofhisH; it shows significant homology with the hisA gene product of E. coli, the sixth gene of the operon encoding the enzyme phosphoribosylformimino-5-amino1-phosphoribosyl-4-imidazolecarboxamide isomerase (EC 5.3.1.16), which catalyzes the fourth step of the biosynthetic pathway consisting of an internal redox reaction (Winkler, 1987). This ORF, when aligned with the E. coli hisA sequence of 245 aa, gives an overall identity of 38% (Fig. 4). Moreover, regions are observed of relatively higher similarity, probably involved in the catalytic function of the enzyme and coinciding with similar homologous regions found between the corresponding proteins orE. coil and of two related methanogenic archaebacteria, Methanococcus

voltae and Methanococcus mnnieUii, which, incidentally, have a 70% A + T-rich genome (Cariomaguo et al., 1988; Cue et al., 1985 and data not shown). Unambiguous identification of the start codon was not possible; in addition to the one indicated in Fig. 3, another AUG codon is present 15 nt upstream and in the same reading frame. These data establish that this ORF is the $. coelicolorcounterpart ofthe enterobacteria hisA gene. In previous work the Streptomyces genes corresponding to E. coli hisH, hisA and hisF were not unambiguously identified and could correspond to any of three genes known as hisC, hisF or hisl (Russi et al., 1973). Piasmid pIJ64 was able to complement a mutation in the hisC gene of S. coelicolor (Table II), which therefore can correspond either to hisH or to hisA, but not to hisF. The last part ofthe sequenced region encodes a truncated putative ORF of 76 aa (ORF2, Fig. 3) which did not show any significant homolo~ with other h/s gene products of enterobacteria. We would like to propose that, following this positive identification of h/s genes that correspond to the different biosynthetic enzymes, the S. coelicolor A3(2) genes will b¢ identified with the same symbol adopted for the best studied system of the enterobacteria. The use of different symbols for the same gene and ofthe same symbol for different genes in different species is extremely confusing. We thus propose to rename the S. coelicoior A3(2) h/s genes as follows: hisD, hisC, hisBd, hisH and hisA instead of hisA, hisG, hisB, hisC or hisF or hisl.

(d) Conclusions We have isolated a genomic region of S. coelicolor A3(2) which contains the his gene cluster. The structural analysis showed that at least five ofthe his genes are tightly clustered (Figs. 2 and 3) and that most of them are probably translationally coupled. Further studies on the expression of this cluster of genes will be required to establish whether they constitute an operon, although some previous data on coregulation of expression suggested this possibility (Carere et al., 1973; Derkos-Sojak et al., 1985; Russi et al., 1973). A final interesting observation is that the relative order of the genes within the cluster has been preserved among the enterobacteria and the Streptomyces, which, in evolutionary terms, have been considered rather distant species (Woese, 1987).

ACKNOWLEDGEMENTS

We thank M.J. Bibb, T. Kieser and D.A. Hopwood for providing strains and plasmids. We also thank M.J. Bibb and the members of our laboratories for helpful suggestions and critical reading of the manuscript.

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