J. Mol. Biol. (1977)

117, 175-193

Restriction Endonuclease Mapping of the Escherichia coli K12 Chromosome in the Vicinity of the ilv Genes GEOFFREY J. CHILD+?, FRANK

HISAKO OHTSUBO~, EIICHI

SONNEIXBERG~

AND MARTIN

OHTSUBO~

FRECNDLICH~

of Biochemistry’ and Microbiology2 State University of New York at 8ton.y Brook Stony Brook, N. Y. 11794, l:.S.A.

Department

(Received 22 February

1977, and in revised form 30 June 1977)

We have determined the EcoRI endonuclease fragment map of the Ah8OdiZv chromosome m well as the EcoRI, Hind111 and Barn1 cleavage patterns of the Escherichia coli chromosome around the ilv genes on F310, F312 and Ah80dilv. Comparison of fragment maps from F-prime ilv DNAs with XhSOdiZv DNA shows the orientation of the bacterial genes on the XhSOdiZw chromosome. These data, taken together with the results obtained by hybridization of ilv messenger RNA to hh8OdiZv DNA digested with strand-specific exonucleases, strongly suggest that the iZwOADGE operon is transcribed from a promoter-proximal iZvE to the ilv0 gene. This is opposite to the direction of transcription previously reported. The nature of the &-dominant operator-like mutations that define iZv0 remains to be clarified. Hybridization data obtained with EcoRI fragments of the ilv gene cluster indicate that these fragments may br valuable for studying transcription of the individual ilv operons. A model for the fusion of parental F-prime factors F’-Zac att*Otrp (F’trp/T) and F-i/e (F16) is proposed in which recombinat’ion and inversion of bacterial DNA between the two copies of the F DNA sequences at, position 2.8F to 8.5F allows with the proposed structurcb. thts formation of a Ah80dilv chromosome

1. Introduction Since it was first discovered that restriction endonucleases make double-stranded breaks at specific sequences in DNA, they have been widely used for dissecting the structure of chromosomal DNAs. Homogeneous DP;A species are cleaved in a highly specific and reproducible manner (reviewed by Nathans & Smith, 1975). Agarose gel electrophoresis on both analytical and preparative scales then represents a highly versatile and sensitive technique for the rapid resolution of cleaved DNA based on the molecular weight of the DNA fragments (Sharp et al., 1973; Helling et al., 1974). The genes contained on these individual fragments represent a limited portion of t,hc cleaved genome. The Escherichia coli K12 genes which code for those enzymes responsible for the biosynthesis of isoleucine and valine are clustered on the E. coli chromosome (Bachmann et al., 1976). Despite this, however, it is believed that they constitute three separat’e and independently regulated operons (Ramakrishnan & Adelberg, 1965a ; t Present address: Department Veterans Administration Hospital,

of Medicine, Stanford Universit)y Palo Alto, Calif. 94304, U.S.A. 175

School

of Medicine,

The

176

G.

J.

CHILDS

ET

AL.

Dwyer & Umbarger, 1968; Arfin et al., 1969). The product of the ilvB gene, acetohydroxy acid synthase, is regulated by the levels of valine and leucine (Umbarger & Freundlich, 1965). Isomoreductase, the product of the ilvC gene, is not controlled by the branched-chain amino acids but is induced by the products of acetohydroxy acid synthase (Arfin et al., 1969). Threonine deaminase (ilvA), dihydroxy acid dehydrase (&ID), and transaminase B (ilvE), constitute a third operon regulated by isoleucine, valine, and leucine (Freundlich et al., 1962). A second acetohydroxy acid synthase has recently been found in E. coli K12 which appears similar to the valine-insensitive enzyme in Salmonella typhimurium. The genetic locus for this enzyme, iluG, has been shown to be located between ilvD and ilvE. Recent experiments indicate that ilvG may be part of the iEvADE operon in E. coli K12 (Favre et al.. 1976). Initial work indicated that repression of the ilv enzymes takes place, to a large degree, at the level of transcription (Childs & Freundlich, 1975; Lo Schiavo et al.. 1975; Childs et al., 1977). In order to assess accurately regulation of the individual RNA products of each operon in vivo and in vitro, it was hoped that endonucleases would be found which could cleave ilv DNA at points which partitioned each operon on a different fragment. In this paper we describe endonuclease EcoRI. Barn1 and Hind111 fragment maps of the region of the E. coli K12 chromosome in the vicinity of the ilv genes contained on F-prime episomes or on the hh80dilv specialized transducing phage genome. F-prime ilv plasmids are particularly useful for determination of the position of endonuclease cutting sites since the position of individual ilv genes on these plasmids has been determined by heteroduplex analysis in the electron microscope (Lee et al., 1974). In addition, well characterized deletions of portions of the ilv gene cluster are contained on otherwise isogenic F-prime plasmid chromosomes (Lee et al., 1974 ; Marsh & Duggan. 1972). The physical maps derived from this study reveal the direction of transcription of the ilvADGE operon as well as implying a model for the formation of the Xh80dilv transducing phage chromosome.

2. Materials and Methods (a) Bacterial strains and media Bacterial strains are described in Table 1. Restriction endonucleases were purified from the following strains. EcoRI from E. co& strain RY13, endo I- gal- carrying fi+ plasmid, was kindly supplied by S. Mickel; Barn1 purified from Bacillus amyloliquefaciens H, donated by C. Mulder. L-broth contained (per liter), 15 g Bacto tryptone, 10 g yeast extract and 5 g NaCl. The minimal medium used was that of Davis & Mingioli (1950) except that citrate was omitted. It was supplemented with 0.5% glucose and 17 L-amino acids each at a concentration of 50 pg/ml (isoleucine, valine and leucine were omitted). (b)

DNA

isolation

Plasmids were prepared from E. coli by the modification of the method of Sharp et al. (1972) described by Ohtsubo et al. (1974a). Phage DNAs were prepared by phenol extraction of phage particles previously purified by CsCl density gradient centrifugation as described by Childs & Freundlich (1975). (c)

Enzyme

assays

EcoRI restriction enzyme was assayed by the method of Greene reaction mixture contained, as final concentrations: 100 miu-Tris*HCl MgCl,, 50 mM-NaCl, and appropriate volumes of enzyme and DNA

et al. (1974). The (pH 7.6), 10 mxvfto give complete

t All

are derivatives

amyloliquifaciens

strains

B.

KFSSO KF600 JE5333 RY13TFI

AB2988 AB2992 CSH43 MI199

Strain

H

(genotype)

(P6)

of E. coli K12

F8

except

--gal?

Phegr

H.

1 strains

genotype

cI857st68h80 cI857st68h80 tcI867st68h80dik

R. nmyZoZiqui;fncierrs

F~~CL~ZVEGDACB~ F312-i1vEGDA.f

F-prime

Bacterial

TABLE

trp,

arg.

gal, end01 Wild-type

gal,

Wild-type

val’

ilvDAC118,

rc(.A,

his,

trp

ntr

his, recA his, recA

ilvEl2, ilvEl2,

nrgG, argG, -

genotype

Chromosome

and reference

Avitabile et al. (1972) Childs t Freundlich (1975) Childs & E’reundlich (1975) Sharp et al. (1972) Greene et al. (1974) Wilson & Young (1975)

Marsh & Duggan (1972) Marsh & Duggrtn (1972) Cold Spring Harbor Labs

Source

178

G. J.

CHILDS

ET

AL.

digestion. Barn1 was assayed in the same manner as EcoRI except KC1 replaced NaCl. Hi&IX was assayed as recommended by Miles Labs. The reaction mixture contained (as final concentrations) 6 m&I-Tris.HCl (pH 7.5), 6 m&l-MgCl,, 50 mM-NaCl, 5 pg bovine serum albumen; and appropriate volumes of enzyme and DNA to give complete digestion. Exonuclease III from E. coli (Miles Labs) was assayed by the method of Richardson et al. (1964). The 5’ exonuclease induced by bacteriophage h was purified by the metllod described by Little (1967). This purified 5’ exonuclease was generously supplied by D. Finnigan. All exonuclease digestions were done at 37°C for 90 min. After digestion, the on 0.3% agarose gels faster than undigested DNA. DNAs reached a discrete size, migrating (d) Preparation

of RNA

and DNA/RNA

The 3[H]uridine-labeled RNA isolation reactions, using E. coli RNA, were performed (e) Preparation

hybridization

and subsequent DNA/RNA hybridizatiotk as described by Childs & Freundlich (1975).

of restriction

endonucleasea

E’coRI was purified from strain RY13RTFl through the DEAE-cellulose step by thtr method of Greene et al. (1974). One ~1 of enzyme digested 4 fig of DNA in 1 h. The enzyme was stored in 0.4 M-NaCl, 10 mm-KPO, (pH 7.2), 0.5 mhf-EDTA, 5.0 mM-2-mercaptoethanol in liquid nitrogen. Barn1 from B. amyloliquifaciens was purified by a modification of the procedure of Wilson & Young (1975), given to us by C. Molder. Three liters of an overnight culture grown in L-broth yielded about 18 g (wet weight) of cells. Cells were resuspended in 20 ml of buffer (10 mm-Tris (pH 7*5), 10 mM-fi-mercaptocthanol) and sonicated 10x 30 s. The lysate was then centrifuged in a Spinco 60 Ti rotor for 45 min at 50,000 revsjmin at 0°C. Three ml of 10% streptomycin sulfate (freshly made and adjusted to pH 7.5) was slowly added to the supernatant. After stirring for 15 min in ice, the precipitate was removed by centrifugation in a Sorvall SS-34 rotor at 10,000 revs/min for 30 min. Enough saturated (NH&SO, (pH 7.5) was added to the supernatant to yield 50% saturation. After 30 min on ice, the precipitate was again cent.rifuged at 10,000 rovs/min in a Sorval SS-34 rotor. This supernatant was adjusted to 800/, (NH,),SOI and again the precipitate was centrifuged after 30 min on ice. The 50 to SOyA precipitate was taken up in 6 ml of 1 mM-EDTA, 7 mM-p-mercaptoethanol), and dialyzed overPEMT, pH 7.5 (10 mM-PO,, night against PEMT (3 x 11). This solution was diluted wit’11 5 ml of PEMT and loaded onto a 0.9 cm x 20 cm phosphocellulose column equilibrated with PEMT, pH 7.5. The column was washed with PEMT, then eluted with a 2 x 65 ml gradient of PEMT and 0.0 11 to 0.8 M KCl. Fractions (4 ml) were collected. The peak of activity eluted at about 0.6 byKCl. Active fractions were pooled, concentrated in sucrose, and stored in liquid nitrogen. Hind111 was purchased from Miles Labs and stored at 20°C’. (f)

Agarose

gel electrophoresis

Agarose slab gels (15 cm x 13 cm x 0.35 cm or 15 cm x 13 cm x 0.25 cm) were used for all analytical gels. Agarose was melted in electrophoresis buffer (prepared from a 10 x concentrated stock solution with a final concentration of 40 mM-Tris-acetate, 50 m&rsodium acetate, 5 mM-EDTA, pH 7.9) by autoclaving for about 3 min. Gels were poured when agarose solutions were at 60°C. Bromophenol blue and glycerol (to 20% glycerol) were added to give a final sample volume of 10 to 60 ~1. Samples with lambda phage DNA were heated to 65°C for 10 min and quenched in ice to separate loosely associated cohesive end fragments. The sample was subjected t’o electrophoresis for 5 min at 100 V and therrafter at 35 V. After about 18 h at room temperature, t’he dye marker was about 4/5 down the gel. Gels were visualized by staining in electrophoresis buffer containing ethidium bromide (2 pg/ml). After about 15 min the stained bands were visualized by fluorescence over long wavelength ultraviolet light (C51 Transilluminator, Ultraviolet Products, San Gabriel, Calif.). Gels were photographed using an orange filter (Tiffen Photar 23A), and measurements were taken from the prints (Polaroid 55 p/n). Preparative slab gels (25 cm x 13 cm x 0.5 cm) were treated in bhe same manner as the analytical gels. One hundred rg of hh80dilv digested with EcoRI was routinely resolved on these gels,

ESl>OSUCLEASE (g) El&on

MAPPING of

DNA

from

OF

THE

preparatioe

ill*

GENES

179

agarose gels

After st,aining with ethidium bromide (0.5 pg/ml) these gels were shielded from fluorescrnt light t,o prevent extensive nicking of the DNA. Bands werp cut out of t)he gpl with a razor blade and homogenized with excess electrophoresis buffer at 0°C in a Dounce homogenizer. The slurry was placed in an SW41 centrifuge tube and allowed to sit in the cold overnight. It, wa,s then centrifuged in a Spinco SW41 rot)or at, 35,000 revs/min for 30 min a,t 5’C. The supernatant was ext)racted with phenol to remove residual agarose and thr aqueous layer precipitated at ~- 2O”Cl with 2.5 vol. ethanol mad? 0.25 x in sodium acetate (pH 5.5). The precipitate was collected by centrifugation in a Spinco SW27 rotor at in IO mnr-Tris.HCl (pH 26,000 revs/min for 45 min at - 5°C. The DNA was dissolved 7.9) in 1 II~M-EDTA and dialyzed against thr same bluffer. (h) Molecdar

length

tleterminatio~r

‘I’llr~ 10 r,ndonuclease EcoKI-generated fragments of l+‘8(P6) DNA, or hDIL’A fragments, were used as standards in estimating molecular lengths of other DNA species in the same gel. The sizes of the 10 M(P6) fragments W~YY’ determined by electron microscopy (E. Ohtsuho et al., manuscript in preparation). Tl~cxcx sizes in has’s Y Ifi3 were 22.1 ; 11.1; 9.23; 7.48: 4.72: 4.53; 2.38; 1.44; 1.30; a,rld 0.38.

3. Results (a) Endonuclease fragment

maps of the ilv genes on F310 and F312

Our general approach for ordering of the endonuclease-generated fragments involved comigration of genetically and physically defined substitutions and deletions of F-prime plasmid DNAs. We have derived the EcoRI (Ohtsubo & Ohtsubo, manuscript in preparation; cited by Skurray et al., 1976), BamI, and HindTIIcleavagemaps of the fertility plasmid F (Ohtsubo et al., manuscript in preparation; Fig. 1). The structures of a number of F’ ilv episomes derived from F14 have been determined by the electron microscope heteroduplex method (Lee et al., 1974). F310 and F312 are simple deletion mutants of F14. While F310 contains the entire ilv gene cluster within its 12% kbt of bacterial DNA, F312 has 8.4 kb of bacterial DNA with a deletion beginning near ilv0 in the ilcC gene (Lee et al., 1974; Fig. 3). Utilizing the co-ordinates of the bacterial ilv genes contained on F310 and F312 (Lee et al., 1974) as well as the co-ordinates of the F sequences in these chromosomes (Ohtsubo et al., 1974a) in most cases enabled us to determine the order and position of the endonuclease cleavage sites within the bacterial genes (Fig. 3). The large EcoRI-derived fragment, X24, found in digests of both F310 and F312 (see Fig. 2) has one terminus at position 4.7F. From the molecular length of X24 the other end of this fragment should be at position 8*1B approximately 300 base-pairs from the site of the deletion in F312 (Fig. 3). Similarly, we know that fragments X26 through X31 must be derived from the &UC and itvB genes and the remainder of the bacterial DNA carried by F310, since fragments X26 through X31 are unique to F310 digests (Fig. 2). These data indicate that EcoRI may be useful for partitioning the individual ilv operons onto separate DNA fragments for use in in vitro studies of ilv operon function. Comparisons of the size patterns of the DNA fragments generated by the digestion of F, IF310 and F312 by Hind111 (Fig. 7) revealed the physical map of the bacterial genes shown in Figure 8. It does not appear that Barn1 can cut the bacterial genes present on F310 or F312 since only one fragment containing bacterial DNA can be detected on agarose gels after Barn1 digestion (Fig. 9). t Abbreviations

used: lrb, 103 bases.

of the

I

3. Born1 map

16

fl5

ff fl

fi

fl

fljfy

fft/ f2

9l*OF

‘ff3!

tt?!flft

1

t

4.7F

fl6

1 14

t

” 14 fll

??

fl2

zw

3*9F

94.5F/OF

frud IS3 fro8

e3.2F

fr0C

-

7

II

f10

J

9.lF

IS2

, fl2

f4

(f5,fl3,f6)

fg ’

f2

f2

113 f14 f9 fib

19.9F le.5F 1 21.2F \+r+Jr?r

15*OF

IS3

‘f71

rt

fe

I

if61 k9

*t, f5

f

40.4F $J

42.9F

\ *-

y+f 10,

I,,[

t

f7

*

w

f3

33.IF +J

32.6F

,-

T~,T~~,T~~,+II’-’

f5

f3

T/J

fl

49.7F

49.5F

Rep. inc

1

t

f3

kr

60.7F

frof

fl

I

f6

I

,

,

65.OF

FIG. 1. Genetic and cndonuclease cleavage maps of the F chromosome. Details will be published elsewhere describing the methods used to derive the order of the EcoRI-generated fragments (Ohtsubo & Ohtsubo, manuscript in preparation; cited by Skurry et al., 1976) and the Hind111 and Barn1 fragment maps (Ohtsubo et al., manuscript in preparation). Co-ordinates labeled with F and B are distances in kilobase units from the selected origins for F and for chromosomal DNA, respectively. For additional definitions of nomenclature see Davidson et al. (1976).

,

ECORI map

I.

F

F sequence

Cleovoqe mcp of

Mop

from

ENDONUCLEASE

MAPPING

OF

ile

THE F310

F312

-

-c --

X24

e =

=

=

ZXI

Xhaod/lv

-A

fll: 112:

2.28 2-32

__-----

ZZZ

113:

I .44

-= /

= -

X26

-

flC

I.25/‘.3

x27

=

-G

fl6f17,

0.8 0.72

X28 x29 x30

I -

-H -

118:

0.38~-

fl9:

0. 18 ___-

=

~/--

X25

181

GENES

-

F

J

-

x31

-

-K

Fra. 2. EcoRI-generated fragments of F, F310, F312 and Ah80diZv DNA. These patterns were derived from electrophoresis in 0.3, 0.7 and 1.6% agarose gels. Heavy bands denote fragments which contain bacterial DNA. Fragments X27, X29 and X31 from F310 correspond to hh8OdiZv fragments G, I and K, respectively. F DNA is denoted in decreasing order of M, by fl to fl9; chromosomal DNA contained in F’ ilu DNA is denoted by X24 to X31 ; phage DNAs aTe denoted in decreasing order of molecular length by letters A to Z.

(b) Structure

of the hh80dilv

chromosome

Recently, a specialized transducing phage containing the entire ilv gene cluster of E. coli K12 has been isolated (Avitabile et al., 1972). The structure of the genome of this phage was of particular interest since it has been used as a probe to detect ilvspecific mRNA in E. coli K12 (Vonder Haar & Umbarger, 1974; Childs & Freundlich, 1975; Lo Schiavo et al., 1975) and in S. typhimurium (Childs et al., 1977). Since the bacterial region on this phage is derived from a deletion mutant of F14 named F16 (Avitabile et al., 1972; Lee et al., 1974) this region should be isogenic with the bacterial genes present on F310 and F312. In addition, the EcoRI endonuclease fragment map of /\DNA is known (Allet et al., 1973; Helling et al., 1974; Thomas & Davis, 1975), and partial EcoRI fragment maps of $80 and hh80 DNA have been published (Helling et al., 1974). We derived an unambiguous EcoRI endonuclease fragment map of the AhSOdilv phage genome by combining the published data on h, hh80, and 480 and the data shown in Figures 2 and 4. The EcoRI-derived fragments B, E and F of hh80dilv (Figs 2 and 4) correspond to the fragments known to be on the right arm of /\DNA (Allet, et al., 1973; Helling et al., 1974; Thomas & Davis, 1975). Furthermore, fragments C and D of Xh80dilv (Fig. 4) correspond to fragments on the left end of the 480 chromosome (Helling et al., 1974). As shown in Figure 4, three bands of hh80 DNA of 7.8, 5, and 0.7 kb are not present in the EcoRI digest of Xh80dilv DNA. Instead, this digest contains four fragments of bacterial DNA of 15, 1.25, 1, and 0.87 kb (Fig. 4). The bacterial fragments produced by EcoRI cleavage of XhSOdiZv DNA were

182

Q.

J.

itvE

F312

ET AL.

CHILDS

i/d

6.98 7.38 ilvD HvA IlVC 21.2F ?




1 f9, I I, l8,8 7.5. 3.6

ilvE

6.98 7.38 IIVG dvD i/VA

4.78 F3lO

>