Construction and expression of hybrid plasmids containing the Escherichia coli glyA gene

Gene, 14 (1981) 6 3 - 7 2 Elsevier/North-Holland Biomedical Press

63

C o n s t r u c t i o n a n d expression o f h y b r i d plasmids containing the Escherichie coil glyA gene (Recombinant DNA; cloning of E. coli DNA; serine transhydroxymethylase biosynthesis; restriction endonuclease mapping)

George V. Stauffer, Michael D. Plamann and Lorraine T. Stauffer Department of Microbiology, UnDersi~ of Iowa, Iowa Oty, IA 52242 (U.S.A.) (Received October 6th, 1980) (Accepted February 5 th, 1981)

SUMMARY

The Escherichia coli glyA gene, encoding serine transhydroxymethylase (STHM), has been cloned in the plasmid vector pACYCI84. The recombinant plasmid (pGS1) contains a 13 kb EcoRl insert. Genetic and biochemical experiments indicate that the region controlling STHM synthesis is present on the insert. Strains beating multicopy plasmid vectors carrying the glyA gene overproduce the enzyme from 17- to 26-fold. The glyA gene was identified on the insert by analyzing a set of plasmids derived from pGSI that carry random insertions of the transposable kanamycin resistance element TnS. Cloning of segments of the original insert into plasmid pBR322 established that a 2.5 kb Sall.Bcll fragment carries the glyA gene. A physical map of this fragment is presented.

INTRODUCTION

The glyA gene product, STHM (EC 2.1.2.1; L-serine: tetrahydrofolate-5,10-hydroxymethyltrans. ferase), is responsible for the interconversion of serine and glycine. This reaction also produces 5,10-methylenetetrahydrofolate, and is a primary contributor of one-carbon fragments in cell metabolism (Mudd and Cantoni, 1964). Because this reaction has a central function in cell metabolism, an understanding of its regulation is important. The basic work on STHM indicates that Rs control Abbreviations: Ap R, ampicilUn-resistant; Cm S, chloramphenicol-sensitive; kb, kilobase pairs; Kn R, kanamycinresistant; SDS, sodium dodecyl sulfate; STHM, sexine transhydroxyrnethyiase; Tc R or Tc S, tetracycline-resistant or -sensitive; [], brackets indicate the plasmid-bearing strains.

is complex, with several compounds (serine, glycine, methionine, thymine, purines, folates) possibly playing a role in controlling glyA gene expression (Taylor et al., 1966; Mansouri et al., 1972; Stauffer et al., 1974; Miller and Newman, 1974; Greene and Radorich, 1975). Other work has shown that methionine regulatory mutants (metK and metJ) have altered regulation of STHM synthesis, suggesting that a common component(s) exists for control of both glycine and methionine biosynthesis 0Vleedel and Pizer, 1974; Greene and Radovich, 1975; Stauffer and Brenchley, 1977). None of these studies, however, identified the actual component(s) important for the regulatory signal, or provided a mechanism of involvement of the regulatory component(s). in order to understand more clearly the structural and regulatory properties of the giycine operon it would be advantageous to clone this region of the

0 378-1119/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press

64 E. coil chromosome onto a plasraid vector and to

characterize it biochemically. This report describes t_he cloning and analysis of DN A carrying the E. coli glyA gene. (Part of this work has been presented previously [M. Plamann, J. Yeggy and G. Stauffer, AbsL Annu. Meet. Am. Soc. Microbiol., 1980, H102, p. 1251.)

appropriate supplements. These cultures were used to inoculate 0.4% glucose minimal media containing appropriate supplements. The inoculum was added to give a reading of less than 10 Klett units. Growth was monitored with a Klett-Summerson colorimeter with a blue no. 42 falter.

(d) Cell extracts MATERIALS AND METHODS

(a) Strains, plasmids, and phage All bacterial strains used are derivatives of E. coli k-12. GS162 is pheA905, araD139, LffacU169, strA, thi. This strain was constructed from MC4100 (Casa~ban, i976)and was obtained from Dr. G. Zurawski. GS245 is pheA 905 , araD139, A/acU169, ~glyA , strA , thi and was derived from GS162. E. coli K-12 strain W3110 was the source of chromosomal DNA. Hasmids p ACVC184 (Chang and Cohen, 1978) and pBR322 (Bolivar et al., 1977) were provided by Dr. G. Zurawski. Purified ~NM816 DNA (Wilson and Murray, 1979), a replacement receptor for the EcoRI system, was a gift from Dr. !. Crawford. ~b221rex:: Tn5 cl857 (Berg et al., 1975) was from Dr. G. Zurawski.

(b) Media The complex medium was LB (Miller, 1972). Minimal medium was the minimal salts medium of Vogel and Bonnet (1956) supplemented with 0.4% glucose. Other supplements were added at the following concentrations: amino acids, 50/zg/ml (serine and glycine were routinely added at 200 btg/ml); purines and pyrimidines, 10/.tg/ml; vitamins, 1/2g/ml. Antibiotics were used at the following concentrations: ampicillin, 25/zg/mi; cMoramphenicol, 25 pg/ml (200/ag/ml for plasmid amplification); tetracycline, 10 /~,/ml; kanamycin, 25 /J~ml. Agar was added at 1.5% to m ~ e either minimal or LB agar. Tryptone broth, tryptone agar, and tryptone top agar are as described (Miller, 1972). (c) Growth Cultures for enzyme assays were grown overnight at 30°C or 37°C in 0.04% glucose minimal media with

A 40 nd volume of cells at a Klett reading of 100 -+ 5 units was chilled on ice, the cells pelleted by centrifugation at 12100 Xg, and washed two times in 0.85% sodium chloride. The cells were stored as frozen pellets for no longer than 48 h. The thawed cells were resuspended in 2.0 nd of 1.0 M phosphate buffer (pH 7.4) and extracts prepared by sonifying 1 time for 5 s with the ES tip (No. 9118) on a Lab.Line Ultratip Labsonic System at a power setting of 80 W. Centrifugation in an eppendoff model 5412 centrifuge (1.5 ml polypropylene tubes) for 5 min in a 4°C cold room removed debris. Extracts were used within I h for enzyme assays.

(e) Enzyme ~mys STHM activity was determined by the method of Taylor and Weissbach (1965) which monitors the conversion of L-[3-t4C]serine to glycine. "l'he remits are averages of two or more assays in which the reactions were determined in triplicate for each assay,

(f) Protein determinations Protein determinations were made by the method of Lowry et al. (1951). (g) Preparation of chromosomal DNA Chromosomal DNA was isolated from 0.75 g of E. coli cells grown in LB to a Klett reading of 200 units. The cel! pellet was gently resuspended in 1.5 ml of 0.15 M NaCl-O.l M EDTA (pH 8.0) containing 4.0 mg of lysozyme, incubated for 15 min at 37°C, and the mixture frozen in a -60°C bath. Then, 12.5 ml of 0.1 M NaCI-O. 1 M Tris (pH 8.0)- 1.0% SDS was added and the mixture thawed at 65°C. After a second freeze-thaw cycle, the mixture was chilled o,1 ice and extracted two times by gently rolling in 0.5 vol. of phenol saturated with TE buffer [10mM Tris

65 (pH 8.0), 1 ram EDTA]. The DNA was precipitated by the addition of 2.5 vols. of 95% ethanol (-20°C), spooled on a glass rod, drained of excess ethanol, and dissolved in 5 ml of TE buffer. Contaminating RNA was digested with RNase (50 pg/ml final concentration) followed by extraction with 0.5 vol. of phenol saturated with TE buffer. The mixture was adjusted to 0.3 M sodium acetate (pH 6.0), and the DNA precipitated by the addition of 2.5 vols. of 95% ethanol. After +2 h at -20°C, the DNA was pelleted by centrifugation (17300Xg, 4°C, 30 rain), washed 2 times with 70% ethanol, dried under vacuum, and dissolved in 10 ml of TE buffer.

(it) Piasmid isolation Plasmid DNA was prepared from chloramphenicol amplified cells (Clewell0 1972) by an SDS lysis procedure followed by ethidium bromide-cesium chloride equilibrium density gradient centrifugation (Guerry et al., 1973; Selker et al., 1977). To ana!yze transformants for plasmid DNA, small quantities of DNA were isolated using the procedure described by Cameron et al. (1977) as modified by Williams et al. (1979).

(i) Transformation and transfection

E. coli strain GS245 was rendered competent and stored by the cryogenic procedure of Morrison (1977). Transformations were as described (Selker et al., 1977). Transfections were carried out by mixing 5-30/zl of phage DNA (0.2-1.0/,tg of DNA)with 100/A of competent cells. After 30 rain on ice, the cells were incubated for 2 min at 42°C, 10 min at 23°C, mixed with 2.5 ml of precooled (48°C) tryptone top agar, poured over a tryptone agar plate, and incubated at 37°C until plaques developed. Phage were elute0 from plaques (Miller, 1972) and transducing phage carrying the glyA gene were detected by their ability to lysogenize and complement an appropriate bacterial host with a deletion of the glyA gene.

(j) Isolation of Tn5 insertions i~sertions of the transposable kanamycin resistance determining dement Tn5 in plasmid pGSI were induced using phage ?~b221 e1857 carrying the transposable element, Tn5 (Berg et al., 1975). Cells bearing

plasmid pGSI were #own at 37°C in tryptone broth plus 0.2% maltose to approx. 5 X 108 cells/m1, collected by centdfugation and concentrated 5-fold in 0.01 M MgSO4. X::Tn5 phage were added to 0.1 ml volumes of cells at a multiplicity of <1, the mixtures incubated for 1 h at 30°C, then spread on LB plates containing kanamycin (25 gg/ml) plus tetracycline (10/~g/ml) and incubated for 24 h at 30°C. Kn R Tc R cells were harvested from the plates and crude plasmid DNA prepared (Guerry et al., 1973). This DNA was used to transform a glyA deletion strain with selection for Kn R Tc R transformants on LB plates plus the antibiotics. Usually 1 to 5 transformants were recovered using plasmid DNA prepared from a single plate of cells.

(k) Restriction and ligation DNA fragments were generated by digesting from 0.5 to 2/zg of DNA in a 25 ~1 volume using the following reaction buffers for each restriction endonuclease: Xhol and SalI, 6 mM Tris-HC1 (pH 7.9), 150 nO.l NaCI, 6 mM MgCI2, 6 mM 8-mercaptoethanoi, Mboll and EcoRll, 10 ram Tris- HC1 (pH 7.4), 6raM KC1, 10 mM MgCI2, 1 mM dithiothreitol, Hindlll, Pvull, and ttinfI, 7 mM Tris" HCI (pH 7.4), 60 mM NaCI, 7 raM MgCI2, 6 mM/3-mercaptoethanol, Sraal, 6 mM Tris. HCI (pH 8.0), 20 raM KCI, 6 mM MgCI2, 6 mM ~-mercaptoethanol, Taql, 6 mM Tris" HCI (pH 7.4), 6 mM NaCI, 6 mM MgCI2, 6 mM /~.mercaptoethanol, 8cll, 6 mM Tris. HC1 (pH 7.4), 75 mM KCI, 10 mM MgCI2, 1 mM dithiothreitol, ttpal, 10 mM Tris" HCI (pH 7.4), 20 mM KCI, 10 mM MgCI~, 1 mM dithiothreitol, EcoRl, 100 mM TrisHCI (pH 7.5), 50 mM NaCI, 5 mM MgCI2. All buffers also contained 100 ;ag/ml bovine serum albumin. Reactions were incubated for 1 h at 37°C (Bell and Taql were incubated at 50°C and 65°C, respectively), heated to 65°C for 5 rain followed by the addition of 5/al of dye mixture containing 10% FicoU, 0.05% bromophenol blue, 0.05% xylene cyanole FF. Ligations were carried out in a 25 gl reaction volume containing 50 mM Tris. HCI (pH 7.8), 10 mM MgCI~, 20 mbl dithiothreitol, 1 mM ATP, 50 /g/ml bovine serum albumin and DNA (0.1 to 0.6 #g/gl). Incubations were at 10 to 15°C for 6 h. (1) Agarose and polyacrylamide gel electrophoresls Electrophoresis of restriction endonuclease digests were performed in horizontal 0.8% agarose gels in 40

66 mM T r i s - 5 m M sodium acetate (ptl 7 . 9 ) - ! mM EDTA buffer containing 0.2 ktg/ml of ethidimn bromide or in vertical 5,q polyacrylantide slab gels in 90 mM Tris-90 mM borate (pH 8.3)-2.5 mM EDTA buffer. Elec~rophoresis was at ! 20 V for agarose gels and 200 V for polyacrylamide gels and continued untii the tracking dyes, bromophenoi blue and xylene cyanole FF, had migrated an appropriate distance. After electrophoresis, the polyacD'lamide stab gels were stained in an e~hidium bromide solution (! btg[ ml) for 30 rain. The gels were placed on a model C-61 transilluminator (Ultra-Violet Products. Inc., San Gabriel, CA), and photographed with a Polaroid MPL-4 system with Polaroid 665, or 667 film and a Tiffen 23A f'dter. Hindlll and EcoRl genera'ed fragments of lambda DNA were used as molecular weight star, dards for agaro~ gels. Taql, Hinfl. and b.coRll generated fragments of plasmid pBR322 were used as mt~ecular weight standards for polyacrylamide gels. (m) Chemicals and enzymes The dI-L-tetrahydrofolate, adenosine 5'-triphosphate, dimedone, lysozyme, ethidium bromide. bovine albumin, agarose, acrylamide, bis-acrylamide, amino acids, purines, pyrimidines, vitamins and antibiotics were from Sigma Chemical Co. (St. Louis, MO); cesium chloride was from Kawecki Berylco Industries, Inc. (New York, NY); ammonium persulfate, TEMbD, bromophenol blue, and xylene cyanole FF were from Bio-Rad Laboratories (Riclmaond, CA); DL-[3-14C]serine was from New England Nuclear (Boston, MA); restriction endonucleases and T4 DNA iigase were from Bethesda Research Laboratories (Rockville, MD) or New England BioLabs (3evefiy, MA). All other chemicals were reagent grade e.nd commerciall.' available.

RESULTS (a) Cloning of the E. coil glyA gene Chromosomal DNA ofE. coli K-12 W3110 (10/.tg) was digested with restriction endonuclease EcoRl and d~e fragments ligated into the single EcoR! site o f the Cm R Tc R plasmid pACYC184 (5 p.g). Plasmids carrym g a functional glyA gene were isolated from the liga-

tion mixture by selecting Tc R Giy ÷ transformants using an b~: coil glvA deletion strain as the recipient for transformation. Since the EcoR! site in plasmid pACYC184 is within the gene for chloramphenicol resistance, transformants were also scored for chloramphenicol sensitivity. One transfom~ant with the appropriate phenotype was purified for further study. Plasmid DNA (designated pGSI) isolated from the Tca Cm ~ Gly ÷ transformant was cleaved into two fragments with restriction endonuclease EcoRl, one corresponding in mobility to linear pACYC184 DNA and the other to a molecule of approx. 13 kb (Fig. 1, lane 3).

(b) Production of STHM in strains containing g l y A ÷ plasmids To determine whether the glyA gene cloned on a multicopy plasmid increases tile production of the glyA gene product, we measured STHM activity in several plasmid strains. Enzyme levels are 17- to 26fold higher in strains containing glyA + plasmids than in an isogenic glyA ÷haploid strain (Table I).

TABLE I

STIIM levels in strains carryingglyA* plasmids a Strain b

GS162 GS245 [pGS 11 GS245 [pGS9] GS245 [pGSI 1] GS245 [pGS27 ] GS245 [pGS29]

Hybrid plasmid derived from plasmid

pACYCi 84 pBR322 pBR322 pACYC! 84 pBR322

STllM activity c

84 (1) 1976 (24) 1907 (23) 1411 (17) 2160 (26) 1663 (20)

a Cells were grown in glucose minimal medium supplemented with phenylalanine and thiamine since all strains contain the pheA905 thi- mutations. Tc or Ap was added for strains cartying hybrid plasmids degved from pACYCI84 or pBR322 respectively. b GS162 is a glyA +haploid strain used as a control, pGS9 and pGS11 were constructed so that the 13-kb EeoR! fragment is inserted in pBR322 in both opposite orientations. c Specific activities are nmol HCHO generated/mg protein/ min. Numbers in parentheses show relative fold increases in STHM activity in plasmid bearing strains compared to the glyA +haploid strain.

67

1

2

3

4

5

6

7

8

Fig. I. Agarose-gel electrophoresis of DNA fragments demonstrating thai plasmids pGS9 and pGSI 1 have the 13-kb l:'coR| insert Jn both opposite orientations. Lane I, h + Hindill. Lane 2, pACYCI84 + EcoRl. Lane 3, pGS1 +EcoRI. Lane 4, pBR322 + EeoRl. Lane 5, pGS9 + EcoRI. Lane 6, pGSI 1 + EcoRi. Lane 7, pGS9 + Sail. Lane 8, pGS1 ! + Sail.

(c) Expression initiates from the glyA promoter Since in plasmid pGS1 the 13-kb EcoR! fragment is inserted in tlle EcoRl site within the cldoramphenicol resistance gene of pACYC184, it is possible that the glyA promoter is not present on the fragment and that glyA gene expression initiates from a plasmid promoter. We test. ; this possibility in the fallowing way. Wilson and Murrzy (1979) have described lambdoid phages that allow the recovery of in vitro recombinants. One such phage vector, ANM816, has two EcoRl restriction endonuclease sites flanking a replaceable segment of the phage chromosome. The 13 kb EcoRI fragment from plasmid pGSI was inserted in XNM816 (MATERIALS AND METHODS) and STHM activity measured on extracts p,epared from hNM816 glyA ÷ lysogens and isogenic glyA ÷ haploid strains. The soecific activities

of the XNM816 glyA ÷ lysogens are nearly identical with those of the control strain grown under conditions that repress or derepress enzyme levels (Table 11), suggesting that the glyA gene is expressed from its own promoter. In addition, we constructed a pair of plasmids, pGS9 and pGSII, that have the 13-kb EcvRI fragment inserted in opposite orientations in the (coRl site of plasmid pBR322. The original plasmid pGSI contains one recognition site for Sall within the 13-kb insert. Plasmid pBR322 also has one recognition site for Sail within the tetracycline gene. The position of the Sail site in the insert relative to the Sail site in pBR322 should be different in the two possible orientations. As predicted, digestion of plasmid pGS9 with Sail yields two fragments of 15.0 kb ~nd 2.4 kb (Fi~. 1. lane 7), while digestion of plasmid pGSI 1 with Sall yields two fragments of 11.9 kb and 5.5 kb (Fig. I, lane 8). Both plasmids

TABLI: II Regalation of STtlM activity m XNM816 glyA* lysogens Additions to glucose minimal medium a

Serine Uanshydroxymethylase activity b GS162

GS245 zsg/yA O,NM816

glyA "

glyA 4) Isolate I

None Triraethoprim (0.25 #g/ml) Serine + glycine + methienine + adenine + g~aanine + thymine

Isolate II

87

85

92

159

163

174

44

54

63

a Concentrations of supplements were as stated in MATERIALS AND METHODS. Phenylalanine and thiamine were included in the media since all strains contain the pheA905, thi- mutations. b Specific activities are nmol HCHO generated/rag protein/ rain. GS162 is a glyA +haploid strain used as a control. Parentheses specify the prophage in the lysogen.

retained the ability to complement the glyA deletion and enzyme assays demonstrated that transformants containing these plasmids have elevated STHM levels (Table I). These results are consistent with the above conclusion that the glyA gene is expressed from its own promoter. (d) Identification of the glyA gene within the 13-kb EcoRl fragment The exFeriments described above demonstrate that the 13.kb EcoRl fragment in plasmid pGS1 contains tl:.e necessary information to direct the synthesis of functional STHM. To locate the glyA gene within the EcoRi fragment, we made use of the transposable kanamycin re,stance determining element TnS. A number of plas:nids were isolated with insertions of the Tn5 element and used to transform a glyA deletion strain with selection for Tc R Kn R transfonnants on LB plates plus the antibiotics (MATERIALS AND METHODS). Transformants were then tested for their ability to grow on glucose minimal plates plus phenylalanine, thiamine, and the antibiotics, with or

without a glycine supplement. Since transposition of the Tn5 element into a gene eliminates gene function (Berg, 1977; Heffron et al., 1977), transformants requiring glycine most likely have the Tn5 element inserted in the glyA gene or its controlling element. The approximate site of each insertion was localized by restriction endonuclease analyses of plasmids bearing the Tn5 element as described below. An EcoRl +Hpal double digest of plasmid pGSI produces fix fragments (Fig. 2A, lane 1). The relative position of each fragment in pGSI is shown in Fig. 2B. Insertion of the Tn5 element should alter the mobility of one fragment. In plasmids pGSi7 and pGS26 for example, fragments 6 and 5 axe altered respectively (Fig. 2A, lanes 2 and 3). Additional fragments in lanes 2 and 3 are due to recognition sites for HpaI in the Tn5 element. Each insertion of Tn5 could be assigned to one of the six fragments produced by EcoRl +Hpal double digests. The Tn5 insertions were more precisely located by digesting plasmid DNA with restriction endonuclease Xhol. Plasmid pGS1 has one recognition site for Xhol (Fig. 2A, lane 5 and Fig. 2C). The Tn5 element has three recognition sites for Xhol, one in the 2500 bp of nonrepeated material and one in each of the 1450 basepair inverted repeats, about 435 bp from either end (Fig. 2C and Rothstein et al., 1980). As predicted, digestion of plasmids bearing the Tn5 element with Xhol produces four fragments (Fig. 2A, lanes 6 and 7). Two of these are from the Tn5 element and were present in all Xhol digests. The remaining two fragments are unique for each Tn5 bearing plasmid and their sizes are dependent on the site of insertion. For example, in plasmid pGSI7, the unique Xhol fragments are about 16.6 and 1.3 kb, while in plasmid pGS26, the unique fragments are about 15.9 and 2.0 kb. The size of either of the unique fragments minus the 435 bp contributed from the Tn5 element def'mes the Tn5 insertion site relative to the single Xhol site in plasmid pGSI (Fig. 2C). The orientation of the two fragments could be assigned since the approximate site of each Tn5 insertion was known from res'alts of the EcoRl +Hpal double digests. Occasionally the two unique fragments were nearly identical in size and additional endonuclease mapping was required to orient the fragments. Data from a number of digests of plasmids carrying random insertions of Tn5 are summarized in Fig. 3 and identify thegl,/A gene within the 13-kb EcoRl insert.

69 A.

1

2

3

4

5

6

7

~Unique pGS17fragment " UniqtJepGS26fragment 2/ 3-4--

!Tn5 fragments

5-

-Unique pGS2fi fragment -Unique pGS17 fragment 6--

,Qs

):,

.

-

-

.

.

.

.

IF.o.,

Fig. 2. Location of Tn5 insertions by endonuclease mapping. (A) Agarose-gelelectrophoresis ofEcoRl + Hpal double digests and Xhol digests of plasmid pGSI end representative plasmids (pGS17 and pGS26) containing Tn5 insertions. Lane 1, pGS1 + EcoRl + HpaI. Lane 2, pGS17 + EcoRi + HpaI. Lane 3, pGS26 + EcoR! + Hpal. Lane 4, 7,+ Hindlll. Lane 5, pGS1 + Xhol. Lane 6, pGS17 + Xhol. Lane 7, pGS26 + Xhol. (13) Physical map of plasmid pGS1 showing the posi*ion of the six fragments produced by anEcoR! + Hpal double digest. The thick line on the plasmid represents the pACYC184 DNA. (C) Schematic diagram showing how the Tn5 insertion in plasmid pGS17 was located relative to the single Xhol site in plasmid pGSI. ~l]le open-box segment represent the Tn5 element. The thick line represents the pACYC184 DNA. The two unique XhoI fragments in pGS17 (Fig. 2A, lane 6), used to located the insertion site, are indicated by double headed arrows.

(e) Subdoning of the glyA gene from pGSl To reduce the size , ,f the initial 13 kb hlsert, plasmid pGSI was partially digested with restriction endonuclease Bell, subjected to ligation, and the DNA used to transform a glyA deletion strain with selection for Gly* Tc R transformants (Fig. 4). The transformants were then screened for the presence o f

plasmid DNA smaller in size that pGS1 (MATERIALS AND METHODS). One plasmid, designated pGS27, has deleted four Bcll fragments and has an estimated size of 8.9 kb (Fig. 4). Next, plasmid pGS27 was digested with restriction endonucleases ..gaff + EcoRI, the fragments ligated into the Sall-EcoRI sites of the Tc R Ap R plasmid pBR322, and the DNA used to transform a glyA deletion strain with selection for Ap R

10

-~nsO "--TT-.. . ~

. . . . .

/-

Gly R transformants (Fig. 4). Since deletion of the Sa/1-EcoRl fragment in pBR322 eliminates the gene for tetracycline resistance, these tranfformants were also Tc S. One Ap s Tc S Gly* plasmid, designated pGS29, is 7.0 kb in size and contains a 3.34 kb SallEcoRl fragment from plasmid pGS27 (Fig. 4).

I

(f) Restriction endonuelease mtalysis of plasmid IK;$29

Fig. 3. Identification of :he g/yA gene in plasmid pGSI. The locations of Tn5 insertions were determined as described in the text and Fig. 2. Symbol + or - after Tn5 indicates that the piasmk~ complements or does not complement a glyA deletion ~rain, respectively. The thick line on the plasmid represents the pACYCi84 DNA. The boundaries of theglyA gene are indicated by the arrow.

pGS I 17kb

_ . ] Sot l " - \

\

~

TC'

u~

Analysis o f single, double, or triple digestions o f plasmid pGS29, and o f purified fragments, allowed us to establish a restriction map of the hybrid plasmid (Fig. 5). Using this technique we have located the following restriction endonuclease recognition sites

Sall, Xhol, Bcll, PruH, Hpal, Sinai, Hinfl, Taql and Mboil.

Bcl I

O/Z

Bcl I {Partial digest) T4 Ligase ¢ Transformation with selection for Gly* Tcr

_~_ / ~ , %

?s~f Sol I ¢ EcoRI 1"4Ligase t Transformation with selection for Gl,' Ap'

Pstz0 pBR3Z2 4.36kb Fig. 4. Schematic diagram of the construction of plasmids pGS27 and pGS29. Thin lines represent E. coil chromosomal DNA. Thick lines represent pACYCI84 or pBR322 DNA. The plasmids and positions of the restriction sites are drawn to scale. All transformations were performed with a glyA-deletionstrain as the recipient.

71 :

~

-

=

_

-

-=_---

:

=tt~

I

3 lll

I I I I

[,

I

I

0

0.5

1.0

,

II

,!

I

1.5

2.0

I

I I

2.5 Kb

Fig. 5. Physical map of the 2.5 kb Sall-Bcli fragment from pGS29 carrying the glyA gone. The positions of the restriction sitesare drawnto scale.

DISCUSSION The initial objective of this study was to clone the glyA gone of E. coil in a plasmid vector. On the basis of several criteria it was shown that the constructed recombinant plasmid carries the glyA gone and its control region: (i) plasmid DNA purified from transformed clones could in turn transform a glyA deletion strain to glycine prototrophy; (ii) strains with multicopy plasmid vectors containing the glyA gone produce from 17 to 26 fold higher levels of STHM than did the control strain of E. coli (Table I); 0ii) the 13-kb EcoRI fragment could be inserted in its two possible orientations in plasmid pBR322 while ~laintaining the ability to complement the glyA deletion, indicating that expression of STHM is not due to read-through from a plasmid promoter; (iv) expression of STHM in the ?~/M816 glyA ÷ lysogens (constructed using the 13 kb ~coR! insert from pGS1) and the control strain of E. coli is nearly identical for several growth conditions (Table I!). Another aspect of this study was to reduce the size of the initial 13-kb EcoRl insert by cloning restriction endonuclease generated fragments carrying the glyA gone into appropriate plasmid vectors. The construction of plasmid pGS29 (Fig. 4) demonstrates that a 2.5-kb Sall-Bcll fragment carries the glyA gone. Evidence from an in v;tro transcription-translation system using pGS29 DNA as template suggests that the E. coli glyA gone codes for a polypeptide of about 46 500 daltons (our unpublished results) and would require about 1.27 kb of DNA. Allowing an additional 0.20 kb for a promoter and terminator region, the Sall.Bcll fragment is still 2bout 1.0 kb larger than needed for ~.heglyA gone. In Salmonella typhimurium, one growth conditioncausing derepression (4-fold) of STHM above the levels observed with cells grown in glucose minimal medium is the addition of 0.5/~g/ml of trimethoprim

(Stauffer et al., 1974). Growth of the control strain of E. coli and the ANMS16 glyA ÷ lysogen~ in the presence of trimethoprim show similar increases in STHM activity (Table II). The addition of trimethoprim to the growth medium should reduce the level of tetrahydrofolate and its derivatives and implicates relates in the regulation of STHM in E. coli. The further characterization of plasmid pGS1 and its derivatives should be useful in understanding the mechanism by which the glycine operon is regulated. A physical map of the glyA gone and its control segment will aid in the isolation of pure DNA fragments for in vitro transcription studies and DNA sequence analysis. In addition, the overproduction of STHM in strains bearing multicopy plasmids carrying the glyA gone should facilitate purification of the gone prL,duct.

ACKNOWLEDGEMENTS This investigation was supported by Public Health Service grant GM26878 from the Natior~fil Institute of General Medical Sciences.

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Commrdcated by D.R. Helimki.