241 Gene, 71 (1988) 247-256

Elsevier

GEN 02595

Cloning and nucleotide sequence of the Streptomyces coeZicoior gene encoding glutamine synthetase

(Recombinant DNA; nitrogen metabolism; gZnA; protein homology; phylogenetic tree)

Lewis V. Wray Jr.* and Susan H. Fisher*

Department of Medical Microbiology and Immunology, University of Kentucky, Lexington, KY 40436 (U.S.A.)

Received 1 March 1988 Accepted 4 May 1988 Received by pubhsher 22 June 1988

SUMMARY

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (gk@ was cloned by complementing the glutamine growth requirement of an Escherikhia coli strain containing a deletion of its gltiLG operon. Expression of the cloned S. coelico[orgId gene in E. colicells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. c~~icolor 2280-bp DNA segment containing the g&A gene was determined and the complete ginA amino acid sequence deduced. Comparison of the derived S. co&color GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.

INTRODUCTION

Streptomyces are an economically important group of Gram-positive bacteria that produce a wide varie- ty of unusual metabolites including antibiotics and other pharmacologically active agents. However, the mechanisms that regulate the production of these compounds are poorly understood. Investigations of

Correspondence to: Dr. S.H. Fisher, at her present address: De- partment of Microbiology, Boston University School of Medi- cine, 80 East Concord Street, Boston MA02118 (U.S.A.) Tel. (617)638-5498. * Present address: Department of Microbiology, Boston Uni- versity School of Medicine, 80 East Concord Street, Boston, MA 02118 (U.S.A.) Tel. (617)638-5498.

Abbreviations: aa, amino acid(s); ADP, adenosine .5’-diphos- phate; Ap, ampicillin; bp, base pair(s); CTAB, hexadexyltrime-

the physiological parameters involved in antibiotic synthesis have shown that their production is often triggered by nutritional limitation. For example, bios~thesis of several ~tibiotics reaches the highest levels during nitrogen-limited growth (Demain et al., 1983).

To better understand the physiology of growth and antibiotic production in these bacteria, we have been investiga~g the regulation of GS, a key enzyme in nitrogen assimilation, in S. coeiicoior. GS regulation is best understood in the Enterobacteriaceae. In

thylammonium bromide; DTT, dithiothreitol; Gin, glutamine- requiring; g&t, GS structural gene; GS, glutamine synthetase; kb, kilobase or 1080 bp; la&, pgalactosidase structural gene; nt, nucleotide(s); Ntr, nitrogen-regulated; ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride; R, resistant; Sm, streptomycin sulfate; YEME, see MATERIALS AND METH- ODS, section b; [ 1, designates plasmid-carrier state.

0378-I 1~9/88/$03.5~ 0 1988 Elsevier Science Publishers B.V. (Biom~~c~ Division)

248

these bacteria GS expression is controlled by the Ntr regulatory system in response to nitrogen availability in the growth medium (Magasanik and Neidhardt, 1987). Interestingly, the Ntr system regulates not only glnA expression, but also the expression of genes involved in dinitrogen futation, amino acid transport and degradation of several amino acids. GS is also regulated at a second level in the Entero- bacteriacaea since GS enzymatic activity can be inactivated in vivo by adenylylation of the GS pro- tein (Reitzer and Magasanik, 1987).

GS regulation in Streptomyces is similar to that seen in enteric bacteria in that GS levels are altered in response to the available nitrogen source (Aharonowitz, 1979; Brana et al., 1986; Paress and Streicher, 1985) and enzymatic activity is modified by adenylylation (Streicher and Tyler, 1981). How- ever, GS is not regulated by adenylylation in all Gram-positive bacteria since there is no evidence for adenylylation of GS in Bacillus subtilis (Deuel et al., 1970; Fisher and Sonenshein, 1984).

To investigate the regulation of GS and to explore the relationship between nitrogen metabolism and antibiotic production in Streptomyces, we have clon- ed and sequenced the glnA gene from S. coelicolor.

Interestingly, the S. coelicolor GS protein has greater similarity to the GS proteins from Gram-negative bacteria than to the GS proteins from other Gram- positive bacteria.

MATERIALS AND METHODS

(a) Bacterial strains and plasmids

E. coli strains used were YMCll (dglnALG) (Backman et al., 1981) JM83 and JMlOl (Yanisch- Perron et al., 1985). Plasmids used for cloning were pUC18 and pUC19 (Yanisch-Perron et al., 1985), and pFW2 (F. Whipple and A.L. Sonenshein, un-

published) which contains a coliphage T7 tran- scriptional terminator (Elliot and Geiduschek, 1984). Standard cloning techniques were used throughout these experiments (Maniatis et al., 1982).

(b) Purification and N-terminal sequencing of the Streptomyces coelicolor GS protein

S. coelicolor strain 51508 (Hopwood et al., 1985) was grown at 32°C to stationary phase in 6 liters of

YEME medium containing 2% glucose (Hopwood et al., 1985). GS was purified by the method of Paress and Streicher (1985) with the following modi- fications. Buffer I contained 20 mM imidazole * HCl pH 7.5, 1 mM MnCl,, 100 mM NaCl, 0.1% j?-mer- captoethanol and 0.12 mg PMSF/ml. After dis- ruption of cells by sonication, l/10 vol. of 10% Sm solution was added and the extract was stirred for 30 min at 4°C. The resulting precipitate was re- moved by centrifugation at 18000 rev./min for 45 min at 4°C in an RC-5B Sorvall centrifuge SS-34 rotor. The supematant was dialyzed against 20 mM imidazole - HCl pH 6.3, 25 mM NaCl, 1 mM MnCl,, and 0.1% jI-mercaptoethanol before At&gel Blue (BioRad) chromatography. A gradient of 1 to 5 mM ADP was used to elute GS from the Al&gel Blue column. Purified GS protein was stored in 50 mM imidazole * HCI pH 7.5, 100 mM NaCl, 1 mM MnCl,, 5 mM DTT and 30% glycerol.

GS eluted from the Al&gel Blue column was > 97 y0 pure and was used directly for N-terminal analysis. N-terminal analysis was performed by Edman degradation using an automated amino acid sequenator.

RESULTS AND DISCUSSION

(a) Cloning of the Streptomyces CoelicolorglnA gene

The S. coelicolor glnA gene was isolated from a plasmid library of S. coelicolor chromosomal DNA by selecting for a hybrid plasmid capable of comple- menting the glutamine growth requirement of E. coli strain YMCll. Since Streptomyces promoters are generally not utilized efficiently in E. coli (Bibb and Cohen, 1982), the library was constructed by in- sertion of S. coelicolor chromosomal DNA down- stream from the E. coli lac promoter in plasmid pUC18. Two plasmids, pSF201 and pSF203 (Fig. l), that were capable of transforming YMCl 1

to Gin’, were found among 6000 ApR transfor- mants. Characterization of the DNA from these plasmids showed that the S. coelicolor chromosomal DNA inserts in both plasmids were almost identical and in the same orientation. The pSF201 plasmid contained a 7.3-kb S. coelicolor chromosomal insert. The pSF203 plasmid contained an additional 200 bp

249

of S. coelicolor chromosomal DNA immediately downstream from the luc promoter (Fig. 1).

Subcloning experiments showed that the Gm+ transforming activity in both plasmids was contained within the S. coelicolor DNA immediately adjacent to the lac promoter. A 2.1-kb Sac1 DNA fragment from pSF201 and a 2.3-kb Sac1 fragment from

Fig. 1. Restriction map and various subclones of the S. coelicolor

glnA region. S. coelicolor A3(2) chromosomal DNA isolated from strain Ml45 (Hopwood et al., 198.5) was partially digested with Mb01 and s~~~~tionated on a sucrose gradient. The E. eoii plasmid library was constructed by ligating 4-8-kb S. coelicolor

chromosomal DNA fragments into the BumHI site of pUCl8, selecting ApR transformants of YMCl 1 and screening for growth on plates containing ammonium and aspartate as the nitrogen source. pSF204 was made by digesting pSF203 with EcoRI + HindHI, ele~~~luting the 7.5-kb DNA fragment containing the gJ& gene, and ligating this DNA fragment into EeoRI + HindHI-digested pUC19. JM83 was transformed with the ligation mixture and the plasmid DNA of ApR Lac - trausfor- mants screened by restriction mapping for the desired construct. To construct pSF206, pSF203 plasmid DNA was digested with SueI, the 2.3-kb Sac1 DNA fragment eleetroehrted, and ligated into the Sac1 site of pUCl9. ApR ~~sfo~~~ of YMCl 1 were se&ted and screened for Gin phenotype. pSF207 was made by digesting pFW2 with HindIII, electroeluting the 500-bp Hind111 DNA fragment containing the T7 transcriptional terminator, and ligating this DNA fragment into the Hind111 site of pSF206. The ApR YMCll transformants were screened for the terminator DNA event by restriction mapping and scored for Gln pheno- type. Abbreviations: ter, transcriptional terminator; ZucP, ZQC

promoter; hatched boxes, S. coeiicolor DNA insert; crosshatched box, coliphage T7 transcriptional terminator DNA fragment; (EamHI/MboI), site of S. coeficolor DNA insertion.

pSF203, e.g., pSF206, complemented the glutamine growth requirement of YMCll (Fig. 1). However, complem~tation was only seen when the S. coelico-

lor DNA fragments were cloned in the same orienta- tion with respect to the Zac promoter as in the paren- tal plasmids. The Gm + complementing activity was further localized to a 1.8-kb segment of S. coelicolot DNA immediately downstream from the fat pro- moter and extending to the SmaI site in pSF203 (Fig. 1) (not shown).

(b) Determination of whether the Streptomyces coelieolor gInA promoter is transcribed in Escheri- chia coli

The observation that complementation of YMCl 1

was dependent on the orientation of the cloned S. m&color DNA fragment suggested that expres- sion of the cloned S, c~~icoIor gld gene in E. coli

cells was dependent on transcription initiating at the iac promoter in pUC18 and pUC19 (compare pSF203 and pSF204, Fig. 1 and Table I). This was confmed by showing that GS expression was abo- lished when a T7 tr~sc~ption~ terminator was cloned between the lac promoter and the S. m&color

TABLE I

Expression of the Streptomycees eoelicolor gld gene in Escherichia

coli cells

Strain” Specific activity b (nmol/min/mg protein)

YMCl 1 <5

YMCl 1 [pSF203] 658 YMCl l]pSF204] <7

YMCl l[pSF206] 585 YMCl l[pSF207] <4

a YMCll cells containing the indicated plasmids (see Fig. 1) were grown in W minimal medium (Bender et al., 1977) con- taining 20 gg Ap/ml and 0.2% glutamine and 0.2% ~o~urn chloride as the nitrogen source. Cells were grown to a Klett of 80 to 100 at 30°C permeabilixed by addition of 1 mg/ml of CTAB, shaken at 30°C for 1 to 2min, and harvested by centrifu- gation at 10000 rev./min in an SS-34 rotor of an RC-5B Sorvall centrifuge. Cells were washed with l/2 vol. of 50 mM imidaxo- le * HCl, pH 7.5,l mM MnCl, and 1 mM DTI, and resuspended in 0.1 vol. of the same buffer for assay. b GS activity was determined by the trausferase assay as de- scribed for S. cuttbya GS (Streicher and Tyler, 1981). The GS activity given is the average of two to three determinations.

250

gh4 gene, e.g., pSF207 (compare pSF206 and pSF207, Fig. 1 and Table I). Similar results were obtained when the T7 transcriptional terminator was subcloned between the lac promoter and the S. coeli- color DNA insert in pSF201 (not shown).

Taken together these results suggest that the ex- pression of the S. coelicolor ghA gene in pSF201,

pSF203 and pSF206 is dependent on the lac pro- moter, and that either the S. coelicolorghd promoter is not transcribed in E. coli cells or the S. coelicolor glnA promoter was not cloned in pSF201 and pSF203. Further experiments have shown that the S. coelicolor gfnA promoter is in fact present in these clones (S.H.F. and L.V.W.Jr., unpublished observa-

GATCCGATTGCTTGCCTGTTGlCCCGAACCCGAACACGGlAClGCGCCCGlAlACGTACCGGGAAAGGCGGGGTGTGCGGGCC 80

CCCGATCCGGCCTAGGCTGGGGAGGGAGCCGGTTAACTTCTGCGAAACAAATGGGTCACGCCCGAGAAATCACCCGTCCC 160

TAGGGTCGAGGAAGCGTGTGCCACCCGCACTGGCCGCACGAACGATCTACCAACCCGGCGGGACGGTCGGGAGTAGGAGG 240

AGCTGG ATG TTC CAG AAC GCC GAC GAC GTC AAG AAG TTC ATC GCG GAC GAG GAC GTC AAG

MET Phc Gln Asn Ala Asp Asp Val Lys Lys Phc Ite Ala Asp Glu Asp Val Lys

300

TTC GTC CAT GTC CGG TTC TGC GAC CTG CCG GGC GTC ATG CAG CAC TTC ACG CTG CCC CCC

Phe Val Asp Val Arg Phe Cys Asp Leu Pro Gly Val net Gln His Phe Thr Leu Pro Ala

360

ACG CCC TTC GAC CCC GAC CCC GAG CAG CCC TTC GAC GGG TCC TCG ATC CGC GGC TTC CAG

Thr Ala Phe Asp Pro Asp Ala Glu Gln Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Gln

420

GCC ATC CAC GAG TCG GAC ATG TCC CTG CCC CCC GAC CTG TCC ACC GCG CCC GTC GAC CCC

Ala Ile His Glu Ser Asp Met Ser Leu Arg Pro Asp Leu Ser Thr Ala Arg Vsl Asp Pro

480

TTC CGC CGG GAC AAG ACC CTC AAC ATC AAC TTC TTC ATC CAC GAC CCC ATC ACG GGC GAG

Phe Arg Arg Asp Lys Thr Leu Asn Ile Asn Phe Phe Ile His Asp Pro Ile Thr Gly Glu

540

CAG TAC TCC CCC GAC CCG CCC AAC GTG GCG AAG AAG GCC GAG GCG TAC CTG GCC TCC ACG

Gln Tyr Ser At-g Asp Pro Arg Asn Vat Ala Lys Lys Ala Glu Ala Tyr Leu Ala Ser Thr

600

CCC ATC CCC GAC ACG GCG TTC TTC GGT CCC GAG CCC GAG TTC TAC GTC TTC GAC TCC GTC

Gly Ile Ala Asp Thr Ala Phe Phe Gly Pro Glu Ala Glu Phe Tyr Val Phe Asp Ser Val

660

CGC TTC CCC ACC CGC GAG AAC GAG TCC TTC TAC CAC ATC GAC TCC GAG CCC GGC GCC TGG

Arg Phe Ala Thr Arg Glu Asn Glu Ser Phe Tyr His Ile Asp Ser Glu Ala Gly Ala Trp

720

AAC ACC GGT GCG CTG GAG GAC AAC CGC GGT TAC AAG GTC CGC TAC AAG GGC GGC TAC TTC

Asn Thr Gly Ala Leu Glu Asp Asn Arg Gly Tyr Lys Val Arg Tyr Lys Gly Gly Tyr Phe

780

CCC GTC CCG CCG GTC GAC CAC TTC GCC GAC CTG CCC GCC GAG ATC TCC CTG GAG CTG GAG

Pro Val Pro Pro Val Asp His Phe Ala Asp Leu Arg Ala Glu Ile Ser Leu Glu Leu Glu

840

CGG TCC GGC CTC CAG GTC GAG CGC CAG CAC CAC GAG GTG GGC ACC GCG CCC CAG CCC GAG

Arg Ser Gly Leu Gln Val Glu Arg Gln His His Glu Vat Gly Thr Ala Cly Gln Ala Glu

900

ATC AAC TAC AAG TTC AAC ACT CTG CTC CCC GCC GCG GAC GAC CTC CAG CTC TTC AAG TAC

Ile Asn Tyr Lye Phe Asn Thr Leu Leu Ala Ala Ala Asp Asp Leu Gln Lcu Phe Lys Tyr

960

ATC GTG AAG AAC GTG GCC TGG AAG AAC GGC AAG ACG GCG ACC TTC ATG CCG AAG CCC ATC

Ile Vat Lys Asn Vat Ala Trp Lys Asn Gly Lys Thr Ala Thr Phe Met Pro Lys Pro Ile

1020

TTC GGT GAC AAC CCC TCG CCC ATG CAC GTC CAC CAG TCG CTG TGG TCG CCC GGC GAG CCG

Phe Gly Asp Asn Gly Ser Gly Met nis Vat His Gln Ser Leu Trp Ser Gly Gly Glu Pro

1080

CTC TfC TAC GAC GAG CAG GGC TAC GCC GGC CTG TCG GAC ACC GCC CGC TAC TAC ATC GGC

Lcu Phc fyr Asp Gtu Gin Gty Tyr Ate Gty Leu Scr Asp lhr Ala Ar@ Tyr Tyr Ile Gly

1140

CCC ATC CTC AAG CAC GCC CCG TCG CTG CTG GCC TTC ACC AAC CCG ACG GTG AAC TCG TAC

Gty 11e Leu Lyr His Ala Pro Ser Leu Leu Ala Phr Thr Asn Pro lhr Vat Asn Ser Tyr

1200

CAC CGC CTG GTC CCG GGC TfC GAG GCG CCG GTG AAC CTG GTG TAC TCG CAG CGC AAC CGC

His Arg Leu Vat Pro GLy Phe Glu Ale Pro Vat Asn Leu Val Tyr Ser Gln Arg Asn Arg

1260

TCG GCC GCG ATC CCC ATC CCG ATC ACG CCC TCG AAC CCC AAG GCC AAC CCC GTC GAG TTC

Ser Ala ALa Ilet Arg Ile Pro Ile Thr GLy Ser Asn Pro Lys Ala Lys Arg Val Glu Phe

1320

CGC GCC CCG GAE GCC TCC GGC AAC CC0 TAC CTG GCG TTC TCG GCG CTG CTG CTG CCC GGC

Arg Ala Pro Asp Ala Ssr Gly Asn Pro Tyr Leu Ala Phe Ser Ala Lcu Leu Leu Ala Gly

1380

CTG GAC GGC ATC AAG AAC AAG ATC GAG CCG GCC GAG CCG ATC GAC AAG GAC CTC TAC GAG

L.eu Asp Glly fle Lys Asn Lys Ile GLu Pro Ala Glu Pro Ile Asp Lys Asp Leu u Glu

1440

CTG GCT CCC GAG GAG CAC GCG AAE GTG GCG CAG GTC CCG ACC TtG CTG GGC GCG GTC CTC

Leu Ata Pro Glu Glu His Al8 Asn Vai At8 Gtn Yak Pro Thr Ser Leu Gly Ala Vst Leu

1500

GAC CGC CTG GAG GCC GAC CAC GAG TTC CTG CTC CAG GGC GAC GTG TTC ACG CCG GAC CTG

Asp Arg Lcu GLu ALa Asp His Glu Phe Leu Leu Gin Gly Asp Vat Phe Thr Pro Asp Leu

1560

ATC GAG ACG TGG ATC GAC TTC AAG CCC GCC AAC GAG ATC GCG CCC CTG CAG CTG CGT CCG

ile Glu Thr Trp fte Asp Phe Lys Ar@ AL8 Asn GLu Ile Ale Pro Leo GLn Lcu Arg Pro

1.620

CAC CCG CAC GAG TTC GAG ATG TAC TTC GAC GTG TGA

His fro His GLu Phe GLu Met Tyr Phe Asp Val *

TCGACGGGTCACGCCGATCCGAC 1679

CGGCGCCCCCGTCCCCGTTTCCCGGGGGCGGGGGCGC~GT~GTGCGTGGCCGAAAAC~~TTCACAGGATGCGGGCGGGCC

>>,>>,*,,,> .,,>> <c<<< <<<<<<*<*<<

GGGATCATGTGTACTGACACGGTTGTTCGAGCCAGGTTGACGCCGGGGGACGGGGAGAGTGCCGGAGATGCCGGATCGCG

1759

1839

ACGACGAGGAGCGCGAGTACGACCTGAGGTGGGCGGAGGGTGC~GAGCACAAGGAGCCCTCGGCGCGCGTCCGGATGCTC 1919

1999

GTCGTGGGTGTCGACCGTCGTGGTGCTCGGCTGTGTGGCCGCGGTGATCGTGCTGCTCGGTTACCTCAACTTCCGGGCGC 2079

CCTACTAGTACCGTCCCGCCCGCTGGTACCGCCCCGCCCGCCCGGGACCGCCGCCCCTCAGCGGAACGCGTCGGCGTTGC 2159

2239

CTCGGGTTGCCGAGGATGTCGAGGGTGTCGTCGGCGAGCTC 2280

251

Fig. 2. Nucleotide sequence of the glnA gene from S. coelicolor. Various restriction fragments of the glnA gene were cloned into the phages Ml3mplg and M13mp19 (Yanisch-Perron et al., 1985). The single-stranded virion DNA was used as a template in the dideoxy ch~-te~~ation sequencing method of Sanger et al. (1977). The dGTP analogues 7-deaza-dGTP (~~usawa et al., 1986) and dITP (Mills and Kramer, 1979) were used to overcome band compressions in the autoradio~aphs. The BIONET (Kristofferson, 1987) computer program GEL was used to assemble the final sequence from the various gel runs. The entire sequence of both strands was determined. The coding strand (5’ to 3’) is shown. The deduced amino acid sequence for GS is shown below the coding sequences. The His residue at aa position 267 (nt positions 1045-1047) and the Tyr residue at aa position 397 (nt positions 1435-1437), which are discussed in RESULTS AND DISCUSSION, section d, are underlined. The inverted repeat at nt 1680-1718 is bighlighted by > and < symbols.

252

tions). Thus, it is unlikely that we would have been able to detect S. coelicolor GS activity in E. coli had we not used a vector that had a properly positioned E. coli promoter. Construction of Streptomyces chro- mosomal libraries downstream from the functional promoter may be a useful strategy for cloning and identifying Streptomyces genes by expression in E. coli cells.

(c) Nucleotide sequence of the gZnA gene

The nucleotide sequence of the 2.3-kb DNA frag- ment encoding GS is presented in Fig. 2. The glnA structural gene is defined by the ORF beginning with the ATG codon at nt position 247 and ending with a TGA translational stop codon at nt position 1654. This ORF would encode a protein of 469 aa with an IU, of 52568. This is in good agreement with the estimated size of GS from SDS-polyacrylamide gel electrophoresis (not shown). The results from se- quential Edman degradation of the first 14 N-termi- nal aa residues of purified GS are in complete agree- ment with the amino acid sequence predicted from the S. coelicolor g1n.A nucleotide sequence starting at the ATG codon.

The G + C content of the DNA sequence is 69% and is comparable to the average G + C composi- tion of Streptomyces DNA (Benigni et al., 1975). As a result of this high G + C content, the codon usage is extremely biased and the third position of the codons from the g1ti structural gene is almost exclu- sively G or C.

The sequence AGGAGG located 6 bp upstream from the translational start codon is complementary to a sequence at the 3’ end of S. lividans 16s RNA (Bibb and Cohen, 1982) and thus is likely to function as the glnA ribosomal binding site in vivo (Shine and Dalgarno, 1976). Immediately downstream from the glnA coding region at nt 1680-1718 is an inverted repeat that could potentially form a hairpin loop in the mRNA. This inverted repeat may be involved in terminating glnA transcription since the S. coelicolor

glnA gene appears to be transcribed primarily as a monocistronic message invivo (S.H.F. and L.V.W.Jr., unpublished observation). Similar se- quences have been shown to have transcriptional termination activity in S. lividans (Deng et al., 1987; Pulido and Jimenez, 1987).

In enteric bacteria, two regulatory genes affecting

the expression of glutamine synthetase are located downstream from the &4 gene (Magasanik and Neidhardt, 1987). To see if analogous regulatory genes lie immediately downstream from the S. coeli-

color glnA gene, the 500 bp of DNA downstream from the g1n.A gene was analyzed for ORFs. Although a number of short ORFs were present, including an incomplete ORF starting at the ATG codon at nt position 2210, none of the deduced amino acid sequences had noticeable similarity with either of the Ntr genes from enteric bacteria. In ad- dition, it appears unlikely that the S. coelicolor glnA

expression is regulated by proteins with DNA binding specificity similar to that of the E. coli ntrA

and ntrC proteins. No nucleotide sequences similar to the ntrA and ntrC DNA consensus sequences (Magasanik and Neidhardt, 1987), were found in the DNA region upstream from the S. coelicolor glnA

gene. Unlike the cloned B. subtilis glnA gene which is

subject to nitrogen regulation in E. coli (Schierer et al., 1985), expression of the cloned S. coelicolor

glnA gene was not regulated in response to nitrogen availability in E. coli cells (not shown). The cloned S. coelicolor GS protein was not adenylylated in YMCl 1 cells (not shown) although the S. coelicolor

GS protein is adenylylated in its natural host (S.H.F., unpublished observations). The latter result is consistent with the observation that the purified S. cattleya GS protein could not be adenylylated by E. coli extracts in vitro (S. Streicher, personal com- munication).

(d) Comparison of Streptomyces coelicolor GS with

GS from other prokaryotes

A comparison of the GS amino acid sequences from S. coelicolor and eight other prokaryotes is shown in Fig. 3. Several features are worth noting. The tyrosine residue at aa position 397 in the S. coelicolor GS protein corresponds to the adenyly- lated tyrosine in E. coli GS (Shapiro and Stadtman, 1968). Since GS activity in S. coelicolor is regulated by adenylylation, (S.H.F., unpublished observation) this residue may also function as the site of adenyl- ation in S. coelicolor.

Also conserved between S. coelicolor and E. coli GS proteins is the region around the histidine residue at S. coelicolor aa position 267. Oxidation of the

SC 1

An 1 111 1

Ab 1

St 1

EC 1

Tf 1

IS 1

CR 1

SC 69

in 71

RI Tl

Ab 71

St 70

EC 70

Tf 72

IS m

CR 70

Se 137

An 139

RI 139

Ab 139

St 13G

EC 13G

Tf 13G

GS $40

cm 139

se zG4

An 210

RI ZOR

Ah ZOS

St 207

EC 207

if 207

es lG3

ca 182

se 275

An 261

RI 27G

Ab27G

St 211)

EC 27G

lf 277

Is 253

CR 252

5c346

An 351

Rl 348

&34G

n3bG

EC 34G

If 347

Gs 324

CR 323

EC 417

An 422 RI 116

Ab 417

St 418

EC 410

If Ll7

RR 393

CA 392

Fig. 3.

253

Alignment of the deduced GS amino acid sequences from S. co&coZor (SC), Anabaenu 7210 (An) (Turner et al., 1983), R. legumi-

nostrum (Rl) (Colonna-Roman0 et al., 1987), A. brudense (Ab) (Bozouklian and Elmerich, 1986), E. coli (EC) (Colon&o and Villafranca, 1986), S. typhimurium (St) (Janson et al., 1986), T. ferrooxiduns (Tf) (Rawlings et al., 1987), B. subtilir (Bs) (Strauch et al., 1988), and C. ocetobutyZicum (Ca) (Janssen et al., 1988). The BIONET (Kristofferson, 1987) computer program IFIND, which is based on the algorithm described by Wilbur and Lipman (1983), was used to align the various GS sequences with one another. These comparisons were then used as a guide to construct the overall alignment. The amino acids are represented by the one-letter code and the sequences are presented from the N- to C-terminal ends. The boxed amino acid residues are identical to the amino acids from S. c~lico~o?.

254

corresponding histidine residue in E. coli GS protein causes inactivation of the enzyme (Farber and Levine, 1986). Interestingly, a crystallographic model for GS (Ahnassy et al., 1986) places this region at the active site of the enzyme.

The most striking aspect of the comparison between the various prokaryotic GS sequences is that GS from S. coelicolor more closely resembles GS from the Gram-negative bacteria than that from two Gram-positive bacteria (B. subtilis and C. aceto-

butylicum). This is most noticeable in the regions from aa residues 76-104 and 386-448. Further- more, to achieve optimal alignment of the sequences, it was necessary to place a large gap in the B. subtilis

and C. acetobutylicum sequences at aa positions 140 and 139, respectively (see also Strauch et al., 1988). It was not necessary to add this large gap to the S. coelicolor sequence.

To demonstrate the degree of similarity between the GS sequences, a phylogenetic tree was con- structed using a distance matrix method (Fitch and Margoliash, 1967). For this analysis difference scores between various sequences were calculated (Feng et al., 1985) using the sequence alignment shown in Fig. 3. This analysis places S. coelicolor

with the Gram-negative bacteria (Fig. 4). In con-

Bacillus subtilis

Clostridium acetobutylicum

i

Streptomyces coelicolor

Anabaena 72 10

Rhizobium leguminosarum

Azospirillum brasilanse

Escherichia coli

Salmonella typhimurium

Thiobacillus ierrooxidans

Fig. 4. Phylogenetic tree derived from analysis of the GS se- quences. Similarity scores between the various GS sequences were calculated using the unitary matrix method described by Feng et al. (1985). The similarity scores were converted to differ- ence scores as described (Feng et al., 1985) except that the ad- justment for random alignment was omitted. These difference scores were used as data for the computer program KITSCH (Felsenstein, 1985). This program constructs phylogenetic trees based on the distance matrix method of Fitch and Margoliash (1967) with the assumption that there is a molecular evolutionary clock. The horizontal axis is proportional to the amount of evolutionary time.

trast, a bacterial phylogeny based upon 16s riboso- mal RNA sequence comparisons (Woese, 1987), places Streptomyces in the same phylum with other Gram-positive bacteria.

Because the distance matrix method combines all of the differences between two sequences into a single score, an implicit assumption is made that all positions within a sequence are changing at the same rate. Since this assumption can affect the results of a phylogenetic analysis, the GS sequences were also analyzed by a parsimony method with the computer

program PROTPARS (Felsenstein, 1985). A parsi- mony analysis treats the various sequence positions individually and searches for a tree with the smallest number of postulated changes. This program only allows amino acid changes that are consistent with the genetic code. The number of changes between two different aa residues at a given position is calcu- lated as the number of nucleotide changes necessary to convert a codon for the first aa into a codon for the second aa. Due to limitations of the program, sequences of only 200 aa residues could be analyzed simultaneously, and thus various regions of the GS sequences were examined separately. Although small differences in the tree topology were obtained depending on the region used for analysis (not shown), S. coelicolor was always placed with the Gram-negative bacteria.

A likely explanation for the different phylogenies obtained using GS protein and 16s ribosomal RNA sequences is that the GS protein is not a good molecular chronometer for the measurement of phy- logenetic distances. It has been noted that incorrect conclusions regarding branching order can occur when functional constraints are not uniform over the entire range of examined species (Woese, 1987). The enzymatic activity of the GS proteins from B. subtilis

(Deuel and Stadtman, 1970; Fisher and Sonenshein, 1984) and C. acetobutylicum (Usdin et al., 1986) are not regulated by adenylylation. In contrast, GS from S. coelicolor, E. coli and S. typhimurium is regulated by adenylylation in their natural hosts (Reitzer and Magasanik, 1987; S.H.F., unpublished observa- tion). In addition, the cloned GS protein from

A. brasilense and T. ferrooxidans is adenylylated in E. coli while the cloned GS protein from R. legumi- nosarum is adenylylated in Klebsiella pneumoniae (Barros et al., 1986; Bazouklian and Elmerich, 1986; Colonna-Romano, 1987). Thus the evolutionary re-

255

strictions for GS in B. subtibk and C. acetobutylicum

might be different than for the GS proteins that are regulated by adenylylation. This effect could easily distort the phylogenetic determination.

Moreover, the possibility that interspecies gene transfer of the glnA gene has occurred cannot be ruled out. If such a process had taken place, then differences in the GS sequences could reflect the fact that the grnA gene underwent evolutionary change in an unrelated bacterium.

Other examples of situations in which protein se- quences have similarities that deviate from their predicted taxonomic relationship have been re- ported. This was observed when the cytochrome c2 sequences from several Rhodospirillum species were compared (Ambler et al., 1979). In addition, the pro- tein sequence of a-amylase from Streptomyces limo-

sus has greater similarity to the a-amylases found in eukaryotic organisms than to a-amylases from other Gram-positive bacteria (Long et al., 1987).

ACKNOWLEDGEMENTS

We would like to thank B. Magasanik, A.L. Sonenshein and F. Whipple for providing plasmids and bacterial strains, J. Felsenstein and G.D.F. Wilson for providing computer programs for phylo- genetic analysis, and E. Kashket and A.L. Sonenshein for reading this manuscript. This work was supported by NIH research grant ROl- AI23 168, an Eli Lilly Life Science Grant, Biomedical Research Support Grant No. RR05374 and the University of Kentucky Medical College Research Fund which supported our initial experiments.

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Communicated by K.F. Chater.