Physical identification of an internal promoter, ilvAp, in the distal portion of the ilvGMEDA operon

255

Gene, 76 (1989) 255-269 Elsevier GEN 02886

Physical identification of an internal promoter, ilvAp, in the distal portion of the iZvGA4EDAoperon alarmone; E. coli

(CAMP; CRP; recombinant DNA; non-coordinate expression; oxygen; a-ketobutyrate; K-12; S. typhimurium; galK reporter gene; transcriptional start point)

John M. Lopes * and Robert P. Lawther Biology Department, Universityof South Carolina, Columbia, SC 29208 (U.S.A.) Received

by G. Wilcox:

Revised:

5 October

Accepted:

10 June 1988

1988

6 October

1988

SUMMARY

It has been previously demonstrated that the ilvGMEDA operon is expressed in vivo from the promoters iZvGp2and iZvEp. An additional internal promoter is identified and designated iZvAp.This internal promoter, which allows independent expression of iZvA,has been analyzed both in vivo and in vitro. Our results indicate that: (1) iZvApexists in both Escherichia coli K-12 and Salmonella typhimurium, as demonstrated by fusion to the gaZK reporter gene; (2) iZvApis located in the distal coding sequence of iZvD; (3) the iZvApsequences are not identical for these two bacterial species; (4) transcription from ilvAp of E. cofi K-12 was demonstrated; (5) expression from iZvApresponds to the availability of oxygen; (6) potential 3’ 5’-cyclic AMP receptor protein binding sites exist adjacent to iZvAp.

The ilvGMEDA operon consists of five genes the products of which are required for the biosynthesis of ILV (Lawther et al., 1987; Cox et al., 1987). The entire operon can be transcribed as a unit from the promoter ilvGp2, upstream from iZvG(Adams et al., 1985; Ortuno and Lawther, 1987; Lawther et al., 1987). Expression of the operon from this site is

regulated by attenuation in response to the levels of aminoacylation of isoleucyl-, leucyl- and valyl-tRNA (Freundlich et al., 1962; Lawther and Hatfield, 1980; Nargang et al., 1980). In addition, expression of the operon is also repressed in response to growth in minimal medium supplemented with these three amino acids (Brenchley and Williams, 1975). Also, it has been demonstrated that there exist internal sites for transcription initiation of this operon in both

Correspondence20: Dr. R.P. Lawther,

Abbreviations:

INTRODUCTION

University

of South

Carolina,

Department

Columbia,

of Biology,

SC 29208

(U.S.A.)

Sciences,

Carnegie

Tel. (803)777-6792. * Current Mellon (U.S.A.)

address:

5’-cyclic bromide;

Department

University,

4400 Filth

of Biological Avenue,

Pittsburgh,

PA 15213

Tel. (412)268-5123.

1989 Elsevier

GalK,

(large)

fragment

polymerase;

Science Publishers

B.V. (Biomedical

ampicillin;

galactokinase;

kb, 1000 bp; LB, Luria Bertani;

designates

0378-l 119/89/%03.50 0

Ap,

Division)

bp, base

AMP; CRP, CAMP receptor

ILV, isoleucine, nt, nucleotide(s);

of E. coli DNA

SDS, sodium plasmid-carrier

polymerase

dodecylsulfate; state.

pair(s);

protein;

CAMP,

3’,

EtdBr, ethidium leucine, valine; PolIk, Klenow I; RNAP,

Tn, transposon;

RNA

[ 1,

256

E. coli K-12 and S. typhimurium. based upon insertion of TnlO

This conclusion is (Berg and Shaw,

1981; Blazey and Burns, 1979), Tn5 (Berg et al., 1979), or Mu (Smith et al., 1976) into the promoterproximal portion of the operon. Specifically, insertions into ilvG and ilvE were only partially polar on expression existence

of the downstream of two promoters

genes, implying designated

the

ilvEp (ex-

pressing

ilvEDA) and ilvDp (expressing

ilvDA). The

location

and molecular

of the inter-

nal promoter,

characterization

ilvEp, has been previously

for both E. coli K-12 (Lopes

sequence

for

extended

using

the

ilvD gene.

constructs

The

fusing

analysis

restriction

ments to the galK gene. The approximate

was frag-

location of

ilvAp was ascertained by transcription in vitro and Sl analysis of cellular RNA. Furthermore, the appropriate region of DNA from S. typhimurium was sequenced to determine the sequence homology of this promoter

as compared

to that of E. coli K-12.

described

and Lawther,

1985;

Wek and Hatfield, 1985) and S. typhimurium (Lopes and Lawther, 1985). However, no further evidence for the presence of ilvDp has been presented. Thus, the significance of the earlier genetic observations for ilvDp is currently unclear. The last gene of the operon, i/VA, encodes threonine deaminase which is the only enzyme that is unique to isoleucine biosynthesis. Threonine deaminase catalyzes the deamination of threonine to yield a-ketobutyrate, and the enzyme is feedbackinhibited by isoleucine (Umbarger, 1956). Several studies (Danchin et al., 1984; Daniel et al., 1983; 1984) indicate that c+ketobutyrate may serve as an alarmone to signal cellular transition from anaerobic to aerobic growth. Their conclusion was based upon observations of both the effects of a-ketobutyrate on cell metabolism and the accumulation of this metabolite upon a shift from anaerobic to aerobic growth (Daniel et al., 1983). Studies conducted in the presence and absence of isoleucine indicated that the ilvA gene product was involved, not the biodegradative isozyme of threonine deaminase (Daniel et al., 1984). Moreover, Daniel and Danchin (1979) demonstrated discoordinate expression between ilvE and ilvA in E. coli K-12 strains mutated in crp, the gene for the CAMP-binding protein. The unique requirement of ilvA for isoleucine biosynthesis and the role of cr-ketobutyrate in an anaerobic to aerobic shift imply the likelihood of a mechanism for the independent expression of iZvA. One possible means for this expression would be an internal promoter upstream from iZvA. This report describes experiments designed to detect and localize promoters upstream from i/VA. Observations with a pBR322 derivative suggested that some expression of iZvA must originate from an internal promoter, ilvAp, located within the coding

MATERIALS

AND METHODS

(a) Bacteria, plasmids and media The bacterial strains used in these studies were CU406[iZvA454, gafT12] described by Baez et al. (1979); FD 1022 [ rb.s-302 : : Tn5, IN(rrnD-rmE) 1, AilvGMEDA724 : : Tn.5-13 1, galK2] constructed in this laboratory; and M152[galK2, recA3, rpsL200, IN (rrnD-rrnE)l] obtained from the E. coli Genetic Stock Center. The plasmids used are described in Table I. LB broth and M63 minimal medium were prepared as described by Miller (1972). Galactose MacConkey agar plates contained 1% galactose and 4% MacConkey base agar (obtained from Difco or BBL). When required, media contained 100 pg Ap/ml. Where indicated, the concentrations of ILV were 0.5, 0.5 and 1.0 mM, respectively. (b) Enzymes and biochemicals Restriction

endonucleases,

T4 DNA ligase, exo-

nuclease III, and PolIk were obtained from New England Biolabs. Sl nuclease was obtained from New England Nuclear Corporation. Calf intestinal alkaline phosphatase was obtained from Boehringer Mannheim Biochemicals. T4 polynucleotide kinase and nucleoside triphosphates were obtained from P-L Biochemicals. [JJ-~~P]ATP was obtained from ICN. D-[ l-14C]galactose was obtained from Amersham Corporation. All other reagents were obtained from Sigma Chemical Company. (c) Anaerobic growth of bacterial cultures Bacteria were grown in liquid or on solid media using standard microbiological techniques. Cultures were grown anaerobically on solid media by placing

251

TABLE

I

Plasmids Plasmid

Reference

Description

pBR322

Cloning

PKD pDR720

galK fusion vector

vector

pRL5

4.5-kb Hind111 fragment,

including

the promoter

(of E. coli K-12) inserted

6.8-kb KpnI fragment 4.9-kb

BarnHI-Sal1

fragment

was derived

portion

of the ilvGMEDA

iZvMEDA (of E. coli K-12), inserted the ilvGMEDA

(Fig. l), including

fragment

Driver

and Lawther

(1985)

into the

This study

LT2),

This study

operon

ilvDA (of S. typhimurium

and Sal1 sites of pBR322. The BarnHI-Sal1

from the bacteriophage

1.3-kb HindIII-Sal1

proximal

into the Hind111 site of pBR322

(Fig. 1), including

inserted between the BamHI pRD129

et al. (1981)

Russell et al. (1984)

KpnI site of pRL5 so as to reconstitute pMT127

et al. (1977)

McKenney

system

galK fusion vector operon

pRLl13

Bolivar

(see Fig. 2)

fragment

. XC. (Taillon et al., 1981)

&-r7. ilvGMEDA

(Fig. 1, ilvD’A’) from pRL113,

inserted

between

the

Driver

et al. (1985)

Hind111 and Sal1 sites of pBR322 pRL137

694-bp

HaeIII-A/u1

leader/attenuator) BamHI pJL189

fragment inserted

from between

and Hind111 synthetic

2.7-kb HindIII-ClaI

fragment

1.3-kb HindIII-Sal1

fragment

630-bp AluI fragment

ilvGp2 and

ilv

Ortuno

and Lawther

(1985)

using

inserted

between

the

This study

inserted

between

the

This study

(Fig. 2)

(Fig. 1, ilvD’A’) from pRD129,

containing

an intact

and Hind111 sites of pK06

linkers

Hind111 and Sal1 sites of pKOl1 pJL230

(containing

the BamHI

(Fig. 1, ilvD’A) from pRL113,

Hind111 and ClaI sites of pBR322 pJL190

pRL5

(Fig. 2)

ilvEp, inserted

between the EcoRI and BamHI

sites

Lopes and Lawther

(1986)

of pK04 pJL291

290-bp HpaII

fragment

EcoRI and BamHI EamHI pJL292

with PolIk)

from pRLl13,

inserted

(tilled-in with PolIk) regenerating

between

the

This study

the EcoRI and

sites (Fig. 2)

480-bp HaeIII of pK04

(filled-in

sites of pK04

fragment

from pMT127 inserted

(filled-in with PolIk) regenerating

between the EcoRI and BamHI the EcoRI

culture plates in an anaerobic jar containing a BBL Microbiological Systems Gaspak. The absence of oxygen was monitored by a BBL Microbiological Systems Gaspak Anaerobic Indicator present in the jar. Anaerobic liquid cultures were grown in culture flasks sealed with rubber stoppers. Each rubber stopper was traversed by a glass tube connected to rubber tubing. Cultures were made anaerobic by evacuating the air from the flask with a vacuum pump (connected to the rubber tubing), refilling with argon and then clamping to inhibit air from re-entering the flask. (d) Recombinant DNA techniques Plasmid DNA was isolated as previously described (Driver and Lawther, 1985). Restriction endonuclease digestions were performed according to the specifications of the supplier. Other recombinant DNA techniques were as described by either

and BamHI

sites

This study

sites

Davis et al. (1980) or Maniatis et al. (1982). DNA restriction fragments were sequenced as described by Maxam and Gilbert (1980). (e) Sl nuclease mapping analysis and galactokinase (GalK) assays RNA was extracted using the procedure described by Jones et al. (1983). The RNA concentration was determined by measuring the absorbance at 260 nm fragments were (40 pg = 1 AX.0 unit). Restriction 5’ end-labeled with T4 polynucleotide kinase and treated with 4000 units of exonuclease III for 30 min at 37°C to generate single-stranded probes (Miller and Sollner-Webb, 1981). The probe and the RNA were coprecipitated by the addition of ethanol, and Sl nuclease analysis was performed as described before (Lopes and Lawther, 1986). GalK was assayed by a method similar to that previously described (Lopes and Lawther, 1986; McKenney

258

et al., 198 1). Protein was determined by the method of Bradford (1976).

RESULTS

sites of pBR322 (Figs. 1 and 2, and Table I). This insert contains approximately half of the coding sequences for the ilvD gene and the entire ilvA gene (Lawther et al., 1987). Ap-resistant pJL189 transformants of CU406 (ilvA454) were found to grow in the absence of isoleucine. It is conceivable that expression of the ilvA gene from this derivative does not originate from ihAp but from the promoter, pl (Fig. 2) adjacent to the Hind111 site in pBR322 (Brosius et al., 1982). This seems unlikely because the distal portion of ilvD (downstream from the Hind111 restriction site) contains a strong polar site (Smith et al., 1976; Driver and Lawther, 1985). The presence of this site would make it unlikely that transcription originating from the pBR322 promoter could result in expression of iZvA.

AND DISCUSSION

(a) Expression of threonine deaminase (ilvA) from ilvAp

The analysis described here was designed to identify and localize an internal promoter, ilv& suggested by the previous studies of Daniel and Danchin (1979). This promoter had escaped detection by the genetic analysis indicating the presence of other potential internal promoters (Berg et al., 1979; Berg and Shaw, 1981; Blazey and Burns, 1979; Smith et al., 1979). However, observations by Daniel and Danchin (1979) indicated that threonine deaminase was apparently differentially expressed during growth shifts from a site downstream from ilvEp. To examine the possibility of a promoter existing upstream from ilvA in E. coli K-12 (ihAp), a pBR322 derivative was constructed. The plasmid pJL189 was constructed by inserting a 2.7-kb HindIII-CluI restriction fragment between the Hind111 and CZaI

e_a

EP

I

I

1

0

VK I I

K -12

S. typhimurium

AP

I

I

I

I

2

3

4

5

S HC I II

VCX I I

AHHH

S

I

I

Fig. 1. Partial restriction attenuator

I

1

I

x I

s 1

K

X

S

I

I

I

I

v - -Pvuii

c - -Ciai

K-ml

-Hind Iii

of E. coli K-12 and S. ryphimurium

ilv genes. The distance sites located

I

X - -Xhoi

ilvGp2 (Gp2), ilvEp (Ep), and ihAp (Ap) are indicated.

the CluI, Hind111 and PvuII restriction

K I

C I

B

H-

(a) and the five structural

I 8

I

B-Ml1

maps of the ilvGMEDA operons

E I

kb

V

S - Sal

WHi

I 7

I 6

E-WRi

A-

promoters:

To facilitate the analysis of ihAp, two restriction fragments were tested for the ability to direct expression of the galK gene. A 1.3-kb HindIII-Sal1 restriction fragment (that includes the ilvDA junction, Fig. 1) from E. coli K-12 was inserted between the Hind111 and Sal1 sites of pKOl1 yielding pJL190

iiv M _

&&

(3~2

E. coii

(b) Expression of the g&K gene, as controlled by the iZvAp promoter of Escherichia coli K-12

within

Also indicated

in kb is measured

LT2. The approximate

are the regions encoding from the iZvGp2transcription

lOO-bp of each other in ilvD.

location

of the three

the ilv leader peptide

(e), the

start point. CHV indicates

259

(s)Digest

(a)Digest

with

wlth HI

(b)lnsert Hli*Il

Fig. 2. Schematic vectors.

representation

The three promoters

are described communication

RESULTS

of the construction

of the plasmids

(Pl, P2 and P3), described

AND

DISCUSSION,

from K. McKenney.

29 1 bp

restrlctlon

section

by Brosius

pJLl89,

pJL190

and pJL291

et al. (1982) for pBR322,

b, and Table I. The structures

from the corresponding

are indicated.

of pK04

Details

and pKOl1

plasmid

of constructions

are from a personal

260

(Fig. 2 and Table I). Also, a 290-bp E&z11 fragment was inserted between the EcoRI and BamHI

sites of

pK04 (regenerating the EcoRI and BamHI sites) to yield pJL291 (Fig. 2 and Table I). Either plasmid transformed

into Ml52

(gulK2) resulted

in colonies

that exhibited a pink phenotype on galactose MacConkey agar (indicating a low level of GalK activity;

see Table IV). Consistent

tion is that the transformants medium

containing

source. The level of GalK

activity

a sole

generated

ing pMT127

carbon

and

TABLE

II

Lawther,

(Taillon et al., 198 1;

1986), the parallel

Specific activity of GalK present in M 152 carrying

study

of

the ilvAp-galK

fusion plasmids Plasmid a

(Promoter/bacteria)b

GalK’

None

-

pK04

-

pJL230

(ilvEp/E.c. )

pJL190

(iZvAp/E.c.)

15.6

pJL291

(ilvAp/E.c. )

21.3

pJL292

(ilvAp/S.t. )

10.7

a Plasmids

are described

0.0 8.8 16.2

in Table I. The host strain (M152) was

grown in M63 minimal medium (Miller, 1972) to approx. 2

x

the i&promoter,

and its bacterial

fused to galK (E.c. is E. coli K- 12 and AND METHODS,

section

that

is

S.t.is S. typhimurium LT2).

c nmol ofgalactose-I-phosphate/min/mg RIALS

source,

of protein (see MATEe).

fragment

and

(that includes

isolating

a

the ilvDA

nucleases and the products analyzed by Southernblot hybridization. Fig. 3A shows the result of an experiment in which the PvuII-BglII restriction fragment from pMT127 was cleaved with Suu3A (lane l), RsaI (lane 2), HpaII (lane 3), HaeIII (lane 4) and AZuI (lane 5). The restriction fragments were then transferred to a solid support (Nytran; Smith et al., 1984) and hybridized with the E. coli K-12 iIvApcontaining 290-bp EcoRI-BamHI restriction fragment (labeled by nick translation) from pJL291 (Fig. 2). Fig. 3B shows the result of the Southernblot hybridization of the S. typhimurium LT2 DNA with the E. coli K-12 probe. One restriction fragment from each digest hybridized to the 290-bp probe, a 690-bp Sau3A fragment, a 450-bp RsaI fragment, a 270-bp HpaII fragment, a 450-bp HaeIII fragment and a 195-bp AZuI fragment. The 450-bp HaeIII fragment was chosen for the subsequent analysis of ilvAp because it was readily purified from other restriction fragments, Also, its size made it both suitable for DNA sequencing, and likely to contain any accessory sequences necessary for expression from ilvAp. The fragment was inserted into pK04 using the EcoRI and BamHI sites to yield pJL292 (Table I). The level of GalK activity generated by this construct is indicated in Table II. Ml52 transformants of pJL292 synthesize 10.7 units of GalK. Thus ilvAp from S. typhimurium appears to be a weaker promoter than iZvAp from E. coli K- 12 (Table II). (d) Sl analysis of cellular RNA

10’

cells/ml. b Indicates

sequences homolo-

junction; Fig. 1). This restriction fragment was subsequently cleaved with a number of restriction endo-

by Ml52

(c) Identification of an ilvAp-containing restriction fragment from Salmonella typhimurium LT2 In our previous investigations

ilvAp from the iIvGMEDA

with PvuII + Bg/II

1.8-kb restriction

transformants grown in minimal glucose medium is indicated in Table II. The GalK activity derived from plasmids pK04 (parental plasmid) and pJL230 (ilvEp-galK fusion) are presented for comparison. Plasmids pJL190 and pJL291, the two E. coli K-12 ilvAp-galK fusions, yielded activities of 15.6 units and 27.3 units (nmol/min/mg protein), respectively. The specific activities of GalK for M 152 bearing the different iZvAp-galK fusions are similar to M 152 containing the ilvEp-galK fusion, pJL230 (16.2 units), indicating that iZvAp serves effectively as a promoter under these conditions.

Lopes

To characterize

operon of S. typhimurium LT2, several restriction fragments generated by digestion of pMT127 (Table I) were analyzed for nucleotide

with this observaas

K-12.

gous to iZvAp of E. coli K- 12 by Southern (1975) blot hybridization. This analysis was conducted by cleav-

also grew on minimal

galactose

homologous genetic elements in S. typhimurium LT2 has facilitated our analysis of these elements in E. coZi

To determine the transcription start point in vivo from iZvAp, cellular RNA was analyzed using Sl nuclease. The strain FD1022, deleted for the ilvGMEDA operon (ilvGMEDA724 : : Tn5) was

261

0

A 1234512345

1353

w

1076

-F

872

310 2611271

-

----t

234

m

----F

-c-

270

3--

175

72 -

Fig. 3. Restriction

digest analysis

HpaII (lane 3), HaeIII with EtdBr

and photographed. with a nick-translated

autoradiograph

is displayed

on the right margin

(Panel A) One pg of pMT127

(lane 5). The products

The locations

in bp). (Panel B) The restriction hybridized

of pMT127.

(lane 4) andAlu1

fragments

(Table I) was digested

were fractionated

of $X174/HaeIII

marker

electrophoretically

restriction

shown in panel A were electrophoretically

290-bp, EcoRI-EamHI

with the lane numbers

fragment

corresponding

from pJL291

fragments

with Sau3A

are indicated

transferred

(lane l), KsaI (lane 2),

on a 5% polyacrylamide onto Nytran

(see Fig. 2) and exposed

to those of panel A. The approximate

gel, stained

on the left margin membrane.

(length

The blot was

to x-ray film. The resulting

length of each band is indicated

(in bp).

transformed with pJL291(Table I; E. coli K-12 i/v@). This insured that products of the S 1 nuclease analysis reflected plasmid-directed transcripts, An EcoRI-BumHI restriction fragment (Fig. 4B, ~291) from pJL291 served as the hybridization probe for the analysis of the cellular transcripts. The results of this analysis are shown in the autoradiograph presented in Fig. 4A. RNA was isolated from FD 1022[ pJL291] hybridized to ~291 and treated with Sl nuclease. A 125nt protected product was observed (Fig. 4A, lanes 1 and 2). Lane 1 contains the products of a reaction which contained 25 pg of RNA while in lane 2,50 pg of RNA were used. The intensity of the protected fragment correlates with the amount of RNA. This

protected fragment was not observed when RNA from FD1022 or tRNA was used (Fig. 4A, lanes 3 and 4, respectively). This latter observation implies that the protected product is plasmid-borne. The length of the protected product suggests that iZvApderived transcription initiation in vivo occurs from the same site that is observed in vitro (not shown). (e) Nucleotide sequence of ilvAp of Escherichia coli K-12 and Salmonella typhimurium The nucleotide

sequence of the distal portion of portion of iZvA is presented in Fig. 5 (Lawther et al., 1987; Cox et al., 1987). The figure is numbered from the transcription start point ilvD and the proximal

262

234

B

HJgII I I

Barn HI

Source

:

Probe

pJL291

:

~291

Fig. 4. S 1 analysis of transcription in vivo from iZvAp. (Panel A) RNA isolated from FD lOZZ(dilvGMEDA724 : :TnS- 131) transformed with pJL291 (lanes 1 and 2) or untransformed (lane 3) was hybridized to the ~291 probe, which was 5’ end-labeled with [y-32P]ATP (using T4 polynucleotide kinase) and treated with exonuclease III, to generate a single-stranded probe (Miller and Sollner-Webb, 19X1), prior to hybridjzation. Lane 4 contains the products of a reaction using tRNA (E. coli B). The resulting DNA : RNA hybrids were digested with S 1 nuclease and the products fractionated on an 8 M urea/8 y0 polyacrylamide gel. The size of the resulting product is indicated on the left margin (125 nt). Lane M is a $X174/HueIII size standard radiolabeled using polynucleotide kinase. The size of specific DNA markers is indicated on the right margin (in nt). (Maps B) Schematic representation of the probe used in the Sl analysis presented in panel A. The probe was named based upon the plasmid from which it was prepared. The approximate location of iZv.Rpand the direction of transcription are indicated (arrow).

for ihAp (indicated by the arrow) that was calculated based upon both transcription in vitro and Sl nuclease analysis (Fig. 4). The presumed transcription start point is 97 bp upstream from the translation initiation site for ilvA. As a further point of reference the XhoI restriction site at 4.9 kb in Fig. 1 is indicated at -36 bp (X”, Fig. 5). The consensus sequence for E. coli a70 promoters (Rosenberg and Court, 1979; Siebenlist et al., 1980; Hawley and McClure, 1983; Mulligan et al., 1984) is

indicated beneath the presumed location of ihAp. As seen in Fig. 5, this segment of nucleotide sequence is highly conserved between E. coli K-12 and S. typhi~~~~~. The low level of tr~sc~ption noted in vitro from ihAp reflects the poor homology with the consensus promoter sequence. Also, the lower expression observed in vivo with fusions utilizing ihAp from S. t~~~i~u~~~ is consistent with the S. t~p~i~u~~~ sequence sharing even less homology with the consensus promoter sequence. Consistent with this was

263

E. coli K-12 S. typh.

-65 -85 -lO!5 CTATCGACATCCCGAACCGTGGCATTCAGTTACAGGTAAG CGATGCCGAA CTGGCGGCGC * l t l t * l l t l l * * * t G AC C A TG T G T CA G T T

E. coli K-12 S. typh.

GTCGTGAAGCGCAGGACGCTCGAGGTGACA AAGCCTGGAC GCCGAAAAATCGTGAACGTC l * l * * l * * CG A T C C C G

Xa

-45

r,

-25

b tcl

TAtAaT

TGACat

cst

65

45

25

E. coli K-12 S. typh.

AGGTCTCCTTTGCCCTGCGTGCTTATGCCAGCCTGGCAAC CAGCGCCGAC * AAAGGCGCGG * * * l * T G c c T G

E. coli K-12 S. typh.

TGCGCGATAAATCGAAACTGGGGGGTTAATAATGGCTGACTCGCAACCCC * l* * TGTCCGGTGC ** * * * * * A TC T GA T A G G T

“Pa

105

85

i!YA start Fig. 5. Comparison transcription

of the ilvAp sequence

coli K-12 and S. typhimurium LT2. The figure is numbered

from E.

start point for ilvAp (short arrow indicates

between E. coli K-12 and S.

start point and direction).

typhimuriumLT2 with the nucleotides

present

The asterisks

from the proposed

indicate the nucleotides

in the latter organism’s

DNA indicated

that are different

beneath

the asterisks.

HpnII site discussed in RESULTS AND DISCUSSION, section b. The sequence marked b is the consensus do promoter, with capital letters indicating those nucleotides that are most highly X”, indicates

the Xhol restriction

site at 4.9 kb (Fig. l), Hp”, indicates

the

conserved.

our inability to detect transcription

in vitro from

S. typhimurium

ilvAp. However, S 1 nuclease analysis of cellular RNA from FD1022 containing the S. typhimurium ilvAp-galK fusions (not shown) is consistent with the E. coli K-12 results. While ilvAp is a weaker promoter than ilvEp in vitro (consistent with analysis both by transcription in vitro and by homology score; Mulligan et al., 1984; Lopes and Lawther, 1986), pJL190 and pJL291 (ilvAp) generate amounts of GalK activity in vivo similar to pJL230 (iZvEp). Since homology score predicts promoter activity in vitro (Mulligan et al., 1984) this observation suggests that iZvAp may require ancillary

transcriptional

factors

absent

from

the

reactions

carried out in vitro. It has recently been

described that known positively regulated promoters have the conserved ‘T’ (at -12 bp) replaced by an ‘A’

(Dairi et al., 1985). That ilvAp has an ‘A’ at -12, is indeed consistent with the idea that additional transcription factors are required for full activity. Alternatively, non-a7’-dependent promoters are utilized in both the heat-shock response (Grossman et al.,

1984) and for the regulation of nitrogen metabolism (Hirschman et al., 1985; Hunt and Magasanik, 1985) in E. coli K-12. It is possible that ilvAp is actually the substrate for an alternative form of RNA polymerase and that it retains sufficient homology to serve as a substrate in vitro for purified RNA polymerase, that presumably only has 0” associated with it. (f) Analysis for regulation directed GalK synthesis

by repression of ifvAp-

Except for the results indicating that a-ketobutyrate could serve as an alarmone (Danchin et al., 1984; Daniel et al., 1983; 1984), there was no reason to expect that ilvA might be expressed separately from the upstream genes in the operon. Particularly, two types of genetic analysis appeared to indicate the absence of an ilvA specific promoter, i.e., various insertion mutations (Berg and Shaw, 1981; Berg et al., 1979; Blazey and Bums, 1979; Smith et al., 1976) and the absence of expression of iZvA in either

264

nonsense or deletion mutations of ilvD (Driver and Lawther, 1985). The exception to the above information was the differential expression of ilvA in crp mutants (Daniel and Danchin, 1979). Therefore, in an effort to understand the role of ilvAp in the overall expression of the ilv operon, experiments were performed to analyze the regulation of this promoter. The ilvGMEDA operon is multivalently regulated by ILV (Lawther and Hatfield, 1980; Nargang et al., 1980) and it is completely repressed by growth in LB broth (Lawther and Hatfield, 1977; Gayda et al., 1980; Subrahmanyam et al., 1980). To determine whether expression from ilvAp responds to either ILV-specific regulation or to the effect of rich medium, ilvAp-mediated GalK expression was assayed from cells grown under these two conditions. To this end, Ml52 transformed with a series of plasmids was grown in minimal medium with or without ILV or in LB broth. For comparative purposes, Ml52 transformed with pK04, the parental vector (McKenney et al., 1981), and pJL230, an ilvEp-gulK fusion (Lopes and Lawther, 1986), were also assayed. The results of the GalK assays are presented in

TABLE

III

Specific

activity

of GalK

in Ml52

harboring

(Promoter/bacteria

Plasmid a

ilvAp-gulK

Table III. As described, when the transformants were grown in minimal media, the two E. cofi K-12 ilvAp-gulK fusions, pJL190 and pJL291, yielded activities of 15.6 and 27.3 units, respectively. These activities are similar to pJL230 (16.2 units) indicating that ilvAp is as active as ilvEp under these conditions. Similarly, pJL292, the S. typhimurium ilvAp-galK fusion, yielded 10.7 units indicating that it serves less effectively as a promoter than its E. coli counterpart, consistent with the observed differences in the nucleotide sequence. As indicated in Table III, the presence of the 3 aa in minimal medium does not affect GalK expression from the vector (pK04) or the plasmid containing the iZvEp-galK fusion (pJL230). Also expression from each of the ilvAp-galK fusions is marginally affected by the presence of the 3 aa, that is, it does not show the five- to eight-fold repression observed with expression from ilvGp2 (M.J. Ortuno and R.P.L., unpublished observations). The effect of rich medium on GalK synthesis was more pronounced. Regardless of the plasmid present in the transformant, GalK activity is reduced to roughly 20% of that observed in cells grown on minimal medium. Because of the effect of growth on LB

fusions GalK

‘)

specific activity’

Mind

1.9 (90)

1.7 (19)

16.1 (99)

2.6 (16)

15.6

13.5 (87)

3.9 (25)

21.3

16.7 (61)

1.1 (28)

8.4 (79)

2.2 (21)

(ilvEp/E.c.)

pJLl90

(ilvAp/E.c. )

pJL29 1

(ilvAp/E. c. )

pJL292

(ilvAp/S.t.)

10.7

I and II.

b See Table II, footnote

b.

’ See Table II, footnote

c.

d As indicated

in Table II, footnote

’ M63 minimal

medium

GalK

a, and MATERIALS

supplemented

AND METHODS,

with 0.5 mM isoleucine

section a, M63 was used as the minimal

and leucine, and 1.0 mM valine. The percentages

medium.

(in parentheses)

were

as follows: activity

observed GalK

r Ml52 transformed

for cultures

activity

observed

with the indicated

grown in M63 + ILV x loo, in M63 plasmids

was grown in LB broth (Miller, 1972). The percentages

as follows: GalK GalK

(%)

8.8

(none)

pJL230

calculated

LB’

(%)

16.2

pK04

il See Tables

Min + ILV”

activity

activity

observed

observed

in LB broth

in M63 minimal

medium

x 100.

(in parentheses)

were calculated

265

medium on galK expression from pK04, it is not possible to designate the observed reduction in GalK activity as necessarily involving ilvEp or ilvAp. However, as discussed above, analysis of the expression of transaminase B (ilvE) and threonine deaminase (ilvA) from the chromosome indicate both of these genes are severely repressed in E. coli K-12 grown in LB medium (Lawther and Hatfield, 1977). (g) Analysis of ilvAp-directed

GalK synthesis in cells

grown anaerobically

As described, the product of threonine deaminase (a-ketobutyrate), may function as an alarmone in coordinating the transition of the cell from an anaerobic to aerobic environment (Daniel et al., 1983; 1984; Danchin et al., 1984). Two isozymes of threonine deaminase are present in E. coli K-12, the products of ilvA and tdc (Goss and Datta, 1985; Datta et al., 1987). The work of Daniel et al. (1984) indicated that the product of ilvA was necessary for the observed alterations of a-ketobutyrate concentration during anaerobic to aerobic shifts. Consequently, a phenotypic assay was performed to determine if ilvAp functions differentially in an anaerobic environment. This involved transforming M 152 with ilvAp-, ilvEp-(pJL230) and ilvGp2-galK fusions (pRL137) and subsequently growing the

TABLE

IV

Phenotypic grown

assay

of Ml52

aerobically

Plasmid

harboring

ilv-gulK fusions

various

and anaerobically

(promoter)”

Colony

color after growth

16 h

pRL137

(ilvGp2)

for: b

36 h

+o,

-02

+o,

-o*

White

White

White

White

pJL230

(ilv&)

Pink

Pink

Pink

Pink

pJL190

(ilvAp)

Pink

Red

Pink

Red

pJL291

(ihAp)

Pink

Red

Pink

Red

pJL292

(ihAp)

White

Pink

Pink

Red

a The E. coli K-12 described

host

strain

b Colonies of M 152 transfected MacConkey

agar containing

or anaerobically METHODS,

was

M152;

the plasmids

are

in Table I.

(-0,)

sections

with the plasmids

were grown on

galactose

either aerobically

( + 0,)

as described

in MATERIALS

AND

a and c.

transformants on MacConkey agar plates (containing galactose) both aerobically and anaerobically. The transformants were scored for the characteristic red color (indicating galactose fermentation) after 16 and 36 h of growth at 37°C. The results are tabulated in Table IV. Clearly galK expression from neither plasmid pRL137 (ivlGp2) nor plasmid pJL230 (ilvEp) was dramatically altered by growth in the absence of oxygen. Transformants of pRL137 yielded white colonies indicative of the minimal production of GalK under these conditions. Although pRL137 contains a relatively strong promoter compared to the internal promoters, it expresses a low level of GalK because it includes the attenuator which terminates at least 95% of the transcription starting from ilvGp2 under these conditions (Adams et al., 1985) i.e., MacConkey agar is very similar to growth on LB broth which results in low expression of the ilvGMEDA operon. Transformants of pJL230 yielded pink colonies (indicative of a weak promoter) regardless of the conditions of growth. Unlike the two promoters described above, expression from the ilvAp-galK fusions respond to growth in the absence of oxygen. M152, containing either of the two E. coli K-12 ilvAp-galK fusions (pJL190 and pJL291), grows aerobically as pink colonies (on galactose MacConkey medium) after 16 h and turns a darker tint of pink after 36 h. However, when grown anaerobically the colonies became red after 16 h. M152, containing the S. typhimurium ilvAp-galK fusion (pJL292) grown aerobically, needs 36 h to produce pink colonies, but when grown anaerobically the colonies were red after 36 h. These results are consistent with ilvAp responding to give an elevated level of gene expression when cells are grown in the absence of oxygen. (h) Effect of growth media and presence/absence oxygen on ilvAp activity

of

To further assess the effect of anaerobic growth on expression from ilvAp, Ml52 transformants were assayed for ilvAp-directed GalK synthesis when grown in either minimal medium or LB broth to late log phase. Cellular extracts were prepared and GalK was assayed. The results of this analysis are presented in Table V. There was no significant difference observed in the amount of GalK activity

266 TABLE

V

Specific activity when grown

of GalK in M 152 harboring

ihAp-gulK

fusions

anaerobically

Plasmid a

physiological conditions necessary for optimal transcription from ilvAp, i.e., anaerobic growth. Our confidence

in concluding

augmented

Medium’

GalK

specific activity’

S. typhimutium,

+o,

-0,

Ratio d

the transcription How

combined

Minimal

15.6

16.7

1.1

physiology

pJL29 1

Minimal

27.3

21.0

0.8

exists, is

of these sequences

with the determination

in of

start point both in vitro and in vivo.

expression

pJL190

that this promoter

by the conservation

of ilvA fits into the anaerobic

of E. coli K-12 remains

to be elucidated.

pJL292

Minimal

10.7

10.3

1.0

pJL190

LB

3.9

8.7

2.2

pJL29 1

LB

7.7

20.6

2.7

The mechanism for the regulation of genes expressed in response to anaerobic growth is believed to be via positive regulation due to binding of a regulatory

pJL292

LB

2.2

6.5

3.0

factor.

a See Table I. ’ See Tables ’ See

II and IV.

Table IV,

METHODS,

section

footnote

b

and

during

MATERIALS

AND

c.

’ Ratio of GalK formed during anaerobic observed

In E. coli K-12

one gene, fnr,

has

been

identified as being essential for the expression of several genes under anaerobic growth conditions

aerobic

growth

growth

(-0,)

to that

( + 0,).

between cells (containing any of the ilvAp-galK fusions) grown aerobically vs. those grown anaerobically in minimal media. However, when these same transformants were grown in LB broth (which is similar in composition to MacConkey agar) a pronounced difference was observed. Anaerobically grown cultures yielded two- to three-fold more GalK activity than cultures grown aerobically.

(i) Conclusions The presence of the promoter ilvAp for the unique expression of ilvA was unexpected, because previous genetic analysis (Smith et al., 1976; Driver and Lawther, 1985) indicated that expression of iZvA required that transcription be started upstream from iZvD. However, the studies of Danchin and colleagues (Daniel and Danchin, 1979; Daniel et al., 1984; Danchin et al., 1984) on a-ketobutyrate as an indicator of a shift from anaerobic to aerobic growth, suggested that ilvA could be expressed independently of the remainder of the operon. Our results demonstrate that a site in the distal portion of ilvD can serve as a promoter for gene expression from a high-copynumber plasmid. The presence of multiple copies may partially explain why our analysis detected iZvAp, while the previous analysis involving single copy (i.e., genomic) expression failed to yield sufficient threonine deaminase activity. Clearly another aspect of expression from this promoter involves the specific

(Spiro and Guest, 1987; Jones and Gunsalus, 1987) while for S. typhimurium, two separate gene products, oxrA (corresponding to fir) and oxrC, appear to regulate the expression of two different sets of genes (Jamieson and Higgins, 1986). Spiro and Guest (1987) have proposed a consensus sequence for the fir gene product (A--TTGA---TATCAAT-A). Our inspection of the sequence in the vicinity of iIvAp fails to indicate the presence of this of the nucleotide sequence. However, inspection sequence (Fig. 5) indicates that centered at -95 bp, there exists a region of 23 bp that is highly conserved between E. coli K-12 and S. typhimurium. This sequence from E. coli K-12 is 86% homologous to the consensus CRP-binding sequence (AA-TGTGA----CA) proposed by deCrombrugghe et al. (1984). A second sequence located between -8 and + 6 bp shows a 95% match to the CRP-binding sequence. Presumably, these sites may explain the observation that ilvA is discoordinately expressed in a strain containing a CAMP-independent CRP mutation (Daniel and Danchin, 1979). Also consistent with the described anaerobic gene expression is the observation of Phillips et al. (1978) of a change in CAMP levels in cells shifting from aerobic to anaerobic growth. This is of further interest because CAMP regulates the iIvBN operon of E. coli K-12 (Freundlich, 1977). Our data also give the second indication (i.e., increased activity from anaerobic growth on ilvAp, vs. ilvEp, under MacConkey agar, Table IV) for differential expression of iZvA relative to the remainder of the operon. Additionally, our results (because GalK was assayed) eliminate any confusion as to whether the activity assayed originates from ilvA or tdc.

261

Our interest in the possible aroused

because

analysis upon

of the dichotomy

indicating

pendent

of ihAp was

between

genetic

of upstream

nucleotide

of ihA. Our expectation

is that

iZvAp in the cell (based

analysis, ments),

upon nucleotide sequence data, and unpublished experi-

the present

is the weakest

iZvGMEDA operon.

of the promoters

D.L.

and

Burns,

R.O.:

Genetic

organization

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Salmonella typhimurium ilvgene cluster. Mol. Gen. Genet. Blazey,

se-

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Blazey,

177

(1979) 1-11.

of ilvA depended

that expression

the transcription

quences, potential

presence

of the

It must be at least an order of

magnitude less effective than dvGp2 which itself is a relatively poor promoter. The intermediate, a-ketobutyrate, is highly toxic to the cells (LaRossa et al., 1987) and yet the work of Danchin and coworkers demonstrates that cells continue to synthesize this compound to significant concentrations under unexpected conditions (Daniel et al., 1983). At least in part, defining the physiological significance of ilvAp clearly depends upon our further understanding the role of a-ketobutyrate in cellular metabolism.

D.L. and Burns,

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ACKNOWLEDGEMENTS

The authors wish to thank Ms. Debra Williams for clerical assistance, and Dr. S. Henry and Dr. W. McClure of CMU for advice and criticism of this manuscript. This research was supported by Public Health Service Grant GM28021 from the National Institutes of Health.

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