Genome mapping and protein coding region identification using bacteriophage Mu

Gene, 99 (1991) l-7 Elsevier

GENE

03942

Genome mapping and protein coding region identification (Recombinant

DNA;

transposition;

phage T7 promoter;

using bacteriophage Mu

gene expression;

cloning)

Eduardo A. Groisman *, Nikos Pagratis * and Malcolm J. Casadaban Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637 (U.S.A.) Received by R.M. Harshey: 14 February Revised: 1 June 1990 Accepted: 14 November 1990

1990

SUMMARY

Transposons such as bacteriophage Mu provide a means to clone bacterial genes as alternatives to using standard recombinant DNA technologies. A DNA-cloning and gene-expressing system has been developed with a bacteriophage Mu (DNA capacity of 38 kb) vector that combines the Mu transposition capabilities and a specialized promoter from bacteriophage T7. Genes cloned with this vector can be identified by transcription in vivo with T7 RNA polymerase and subsequent host translation. This system, illustrated with the characterization of a 35kb region of the Escherichia coli K-12 chromosome, is applicable to other Enterobacteriaceae, which are hosts for Mu phage, and is potentially applicable to other bacteria, including Pseudomonas aeruginosa, which have Mu-like phage, and to other organisms for which high-frequency transposons are available.

INTRODUCTION

The study of a variety of biological problems often relies on the availability of cloned DNA sequences and/or gene products. A current goal in biology is to map and sequence

Correspondenceto: Dr. M. Casadaban, versity

of Chicago,

920

Tel. (312)702-1074; * Present

Department St.,

Chicago,

(E.A.G.)

University

Department

Technology

Park,

Tel. (312)226-6500.

Abbreviations:

aa, amino

acid(s);

side; kb, kilobase

or 1000 bp; Km, kanamycin;

resistance/resistant;

PAGE,

IPTG,

plasmid-carrier

Nm, neomycin;

0 1991 Elsevier

ORF,

electrophoresis;

R,

sulfate; TCA, trichloroacetic

( ), prophage;

phosphate;

[ 1,

: :, novel joint (fusion,

insertion).

0378-l 119/91/$03.50

IL

Apt, aminoglycoside

XP, 5-bromo-4-chloro-3-indolyl state;

Inc.,

Dr., Chicago,

isopropyl-/?-D-thiogalacto-

polyacrylamide-gel

SDS, sodium dodecyl

acid; Tn, transposon; denotes

Park

Ap, ampicillin;

bp, base pair(s);

frame;

Microbiology,

(N.P.) ThermoGen,

phosphotransferase; reading

(U.S.A.)

660 South Euclid Ave., St.

2201 W. Campbell

60612 (U.S.A.)

open

The Uni-

IL 60637

of Molecular

School of Medicine,

Louis, MO 63110 (U.S.A.) Tel. (314)362-3692; Chicago

of MGCB,

Fax (312)702-3172.

addresses:

Washington

E. 58th

Science Publishers

B.V

the complete genomes of a variety of organisms. The realization of this goal depends on technologies that shorten the time necessary for the manipulation and analysis of DNA. DNA cloning is an important step in this and other studies since it facilitates the overproduction of encoded gene products put under the control of selective gene expression systems (McKenney et al., 1982; Shatzman et al., 1983). An alternative to in vitro DNA cloning systems is the use of transposons to clone genes (Berg et al., 1989). Current schemes are based on derivatives of Mu and related bacteriophages because of their high transposition frequency, near-random insertion specificity, packaging properties, and broad host range (Pato, 1989; Symonds et al., 1987). Several bacteriophage derivatives have been constructed and used for the construction of plasmid (Groisman and Casadaban, 1986; Groisman et al., 1984) or cosmid libraries (Gramajo and de Mendoza, 1987; Groisman and Casadaban, 1987b) from several members of the family Enterobacteriaceae (Groisman and Casadaban, 1987a) and from P. aeruginosa (Darzins and Casadaban, 1989). These libraries can be selected in any bacterial species which is

2 sensitive to the phage used, in this way obviating the need for E. coli as an intermediate host (Darzins and Casadaban, 1989; Groisman and Casadaban, 1987a).

RESULTS AND DISCUSSION

(a) The mini-Mu element and cloning The mini-Mu cloning element Mud5294 was constructed with a ColEl-type replicon and a phage T7 promoter which

In this report, we present a system for both cloning and identification of protein-coding regions. We have incorporated a specialized phage T7 promoter into a mini-Mu

allows transcription by the T7 RNA polymerase to initiate in the vector and read across 117 bp of the Mu right end

replicon element so that transcription with the T7 RNA polymerase can initiate from the plasmid clones and be followed by host translation. This allows the expression and identification of genes in the cloned DNA without a

into the cloned DNA (Fig. 1). The scheme used to prepare gene libraries and express the cloned genes is presented in Fig. 2. This scheme was tested by preparing Mu lysates by

background

thermoinduction

of most host proteins

(Studier

and Moffatt,

by infection

1986; Tabor and Richardson, 1985). We have used this system to characterize physically and genetically the region around

minute

of MC1040-2[pEG5294]

of the AproC recipient

cells followed

XPh43Mucts{F-

A-

(argF-lacIPOZYA)205 trpA(brnQ phoA proC phoB phoR)roisman et al., 1984). Clones containing 24 (Mutts)} (G

9 of the E. coli chromosome.

C

ner

pT7/T3-18 pMB1

C

ner A

rep pMB1

ner A

c

Fig. 1. Construction promoter

sequence

isolated

as described

to Pvull

of the mini-Mu

H

P

1.0

1.7

plasmids.

by Kupersztoch plasmid

The mini-Mu

(1982). The ligation mixture

and Helinski

pT7/T3-18

=

pBR322

Y

pT7T3-18

KmR PT,

pMB1

element

Mud5294

Research

pEG5086

Laboratories,

the Afac strain MC1040-2

et al., 1984) to Km (40 pg/ml) and Ap (25 pg/ml) resistance.

was constructed

into the mini-Mu replicon

(1973), from plasmid

(Bethesda

was used to transform

Tn5

l(F EHGPMNEMB 6.3 7.6 6.6

(5’-TAATACGACTCACTATAGGGAGA)

+ BamHl-cut

Mu

0

+-_)

rep

B

-

(pBC0

I\ 7.7

7.8

the consensus

(Groisman

phage

and Casadaban,

T7 class-111 1986). DNA,

: : Mud15086) was digested with Smnl + BamHl MD) DNA. Molecular

araD 169 araB

Media and general bacterial

I

by incorporating

element Mud15086

Gaithersburg, {F-

I

protocols

: : Mutts A(laclPOZY)74

genetic techniques

and ligated

are from Maniatis

et al.

galU galK rpsL} (Castilho

have been described

by Miller (1972). Plasmid

pEG5086 confers a weak Lac + phenotype to a Alac strain so transformants that showed a Lac - phenotype in lactose MacConkey indicator media were chosen as candidates for having replaced the lac DNA present in the Smal-BumHI fragment by the phage T7 promoter sequences. One transformant harbored

plasmid

pEG5294

with the mini-Mu

element

Mud5294,

as confirmed

by EcoRl,

Pvull,

BarnHI,

Hindlll,

and Kpnl

digestion

and gel

electrophoresis. The Mud5294 element has approx. 4.37-kb from the Mu left end which includes the Mu repressor c, the regulator ner, and the transposition-replication A and B genes, and also has 117 bp from the right end of Mu. This mini-Mu element also contains the TnS-derived uph (KmR) gene and the ColEl-type G, Bglll;

H, ffindlll;

replicon from the pMB 1 plasmid. The arrows on the top ofthe genes indicate M, Smal;

P, Pstl; S, Safl; V, Pvull.

the direction

of transcription.

B, BarnHI;

E, EcoRl;

Helper Mu Phage Mini-Mu replicon

gene x

Induction for Mu intracellular transposition and packaging of mini-Mu generated structures

ti

gene x

J

Infection of Mu lysogenic recipient cells

gene x

gene x

Recombination between Mu homologous sequences in recipient cells

Rifamoicin

Mini-Mu plasmid clone of gene x

IPTG Induction of the lactJV5 promoter controlling expression of phage T7 RNA polymerase gene

-

Inhibition of host transcription

Strand-specific transcription of mini-Mu plasmid clone genes by T7 RNA polymerase

gene x

Helper Mu Phage Fig. 2. Cloning and expression described carrying

by Groisman

et al.

the temperature

scheme. (Top) In vivo cloning with mini-Mu replicon

(1986).Heat induction

of a bacterial

sensitive allele of the repressor

elements.

strain doubly lysogenic

results in intracellular

replicative

Procedures

for preparing

for a mini-Mu transposition

replicon

and using Mu lysates have been

element

of both mini-Mu

and a helper Mu prophage

and Mu and phage lytic growth.

The helper phage can complement the mini-Mu replicon for the morphogenic functions and package by a headful mechanism DNA structures generated by the mini-Mu replicon. Some of the phage particles carry bacterial nt sequences (e.g., gene x) ‘sandwiched’ between similarly oriented mini-Mu elements. Infection

of a recombination-proficient,

(e.g., gene x). The plasmid protein

products,

A Mu lysogenic

lacUV5 promoter by the addition Strand obtained

Mu-lysogenic

clones obtained

is transformed

recipient

results in the generation

are similar to those generated

strain harboring

a defective

with a plasmid clone obtained

I prophage

transcription

after host translation

is achieved

using radioactive

and Moffatt,

by the high specificity

aa can be visualized

plasmid

molecules

harboring

(Bottom) In vivo expression

with the IucI gene and the T7 RNA polymerase

with the mini-Mu Mud5294.

of an inducer of the lac operon such as IPTG (Studier

and plasmid-specific

of circular

by in vitro methods.

Production

by SDS-PAGE

character

and fluorography.

nt sequences

and strand

gene under the control

of the T7 RNA polymerase

1986). Host transcription

and rifampicin-resistant

particular

of plasmid

specific of the

can be accomplished

can be blocked by the addition of the T7 RNA polymerase.

of rifampicin. The products

4 the proC gene, selected as KmR transductants able to grow in the absence of added proline, were obtained at a

overlapping

frequency of 7.7 x 10 _ 7 colonies per plaque-forming unit of the helper Mu phage as previously described (Groisman and Casadaban,

1986). Eight of twelve independent

fragment

which was shared

by the different

plasmid clones. This approach avoids the complex and time consuming steps of subcloning and construction of deletions.

ProC + (b) Protein identification

clones examined also contained the phoA gene as seen by their hydrolysis of the alkaline phosphatase substrate XP (Sigma, 50 pg/ml) in the media (Groisman and Casadaban,

To express

the products

of the different

plasmids,

we

1986). Plasmid DNA was extracted from these clones and analyzed by restriction enzyme digestion using HindIII,

transformed strain BL21(DE3)(MuhPl) (Fig. 4) with DNA from the twelve ProC’ clones by selecting for Km resistance (Makino et al., 1986b). This strain harbors a

BumHI, and Hind111 + BarnHI and gel electrophoresis. This enabled us to construct a map of the region, to determine the relative orientation of the cloned chromosomal

defective I prophage with the E. coli lucl gene and the T7 polymerase gene under the control of the lacUV5 promoter. Addition of inducers of the lac operon such as IPTG results

DNA with respect to the T7 promoter sequences (Fig. 3) and to determine the location of new ORFs by correlating the physical map with the bands observed in protein gels (Fig. 4). The map (Fig. 3), which spans a region of approx. 35 kb of the E. co/ichromosome, is consistent with the published nt sequences ofthe proC(Deutch et al., 1982), phoA (Chang et al., 1986), pholl (Makino et al., 1986a), phoR (Makino et al., 1986b), aroL, and aroM (DeFeyter et al., 1986)genes, and restriction maps of the region (Groisman, 1986; Kohara et al., 1987; Lloyd and Buckman, 1985; Wanner and Chang, 1987). Five ProC + clones (1, 7, 9, 11 and 12) had the mini-Mu Mud5294 in the orientation shown in Fig. 2. The other seven clones had the mini-Mu element in the opposite orientation. The proC gene could easily be located to a 3.5-kb fragment by comparing the minimum

in expression of the T7 RNA polymerase (Fig. 2) and subsequent expression of plasmid genes that are in the same orientation as the T7 promoter, both of cloned and vector origin (Fig. 4). The control lane, originating from plasmid pEG5294, showed bands of approx. 70,33,29, and 8-kDa, which were the sizes expected for the phage Mu A and B proteins, the TnS-derived Km-Nm Apt, and Mu ner gene product, respectively. No bands were seen for the Mu repressor, or the /?-lactamase (on the parental plasmid) which are transcribed from the opposite strand. The MuB gene product was present in amounts several times that of the MuA protein. Our data support the posttranscriptional regulation of the MuA and MuB proteins obtained using transcripts originating from the Mu early promoter (Parsons and Harshey, 1988). Several loci have been mapped to the 9-min region of the

37a

66kDa

-.-

sbmA

phoA

,I

406.1 407.2

I B kb: 397.0

I H 398.6

I I H H 401.7 402.7

proC

phoHK

sbcC

412.7 II HH 410.9 411.0

P 405.6

oroLM

428.8

418.2 419.1 420.0 I B 419.0

I B 416.2

I

r B 437.9

43H2.9

II

3

Fig. 3. Physical

and genetic map of the 9-min region of the E. coli chromosome.

at the ends of the lines indicate

the orientation

of the mini-Mu

replicon

Lines indicate

Mud5294

the DNA present

(as in Fig. 2) with respect

in each of the ProC’

to the bacterial

clones. Arrows

DNA. Clones

1, 7, 9, 11,

and 12 have the mini-Mu in the ( + ) orientation (as illustrated in the lower part of Fig. 1). Clones 2,3,4,5,6, 8, and 10 have the mini-Mu in the opposite ( - ) orientation. The start ofthe ORFs is located within the regions delimited by the wavy lines (near the top). The arrows above the protein sizes indicate transcription 1987).

orientation

of their respective

genes. Coordinates

(kb) are presented

relative to the published

E. coli K-12 restriction

map (Kohara

et al.,

Clone*

1

IPTG

-

2 +

-

3 +

-

5

4 +

-

6

PEG5294

7

ClOrEX

+-+-+-+-+

IPTG

8 -

9

10

11

12

PEG5294

+-+-+-+-+-+

46.0 -

-

MuA

-

PhoA

> MuB Apt i Proc y AroM 163-

ii

Ir 123-

183-

- AroL

-

-AroL

123-

Ner

- Ner

Fig. 4. Products translated

of the ProC + plasmid

by the host was carried

phage I derivative

harboring

1986). Recombinants After overnight

a final concentration tubes containing

coli BF-

the different mini-Mu

proC+

under the control

clones were grown overnight

incubation

4 ~1 of ‘H-labeled

at 30°C for 30 min, 6 ~1 of rifampicin was carried

analysis,

0.1 y0 SDS-15%

was precipitated

aa mix (Amersham

(Sigma) solution

left and the names

defective)

of the lacUV5 promoter

and

gal]. Phage DE3 is a (Studier

(50 mg/ml in N,N’-dimethylformamide)

37 MBq, 1 mCi/ml) and the incubation

and Moffatt, Km.

PAGE was performed

current

with 5-~1 samples

per well according

per gel. At the end of the run the gels were impregnated

for another

were precipitated to Laemmli

of the proteins

identified

on ice for 10 min and collected

by spinning

buffer. For protein

(1970). The gels were run at room temperature

with EN’HANCE

(New England to Kodak

shown here), and one week. The sizes (kDa) of the unlabeled

are indicated

to eppendorf

20 min at 30°C. At the end of the

in 30 ~1 of 1 x Laemmli cracking

the gels in water for 30 min. The gels were dried for 2 h at 60°C and exposed

times: 8 h, three days (which is

was added to achieve

from each culture were transferred

continued

The pellets were washed with 1 ml of cold acetone and finally resuspended

by incubating

for three different

by the T7 RNA polymerase

at 30°C in 2 ml of M9 glucose minimal media containing

out at 30°C for 20 min when 250~~1 aliquots

labeling period the cells were lysed by adding 43.75 ~1 of 50% TCA, the labeled proteins

3 h at 40 mA constant

transcribed

and modification

were diluted to a final volume of 5 ml and A 6oo = 0.1. They were incubated at 30°C for 3 h (A,,, between 0.350 of them at 0.400) when 1.5 ml was taken from each culture to a clean tube and IPTG was added to a final concentration

of 200 pg/ml. Incubation

for 5 min in the microfuge.

products

hsdS (restriction

the cultures

and 0.690 with the majority of 1 mM. After further

labeling of clone-specific

the phage T7 gene 1, coding for the T7 RNA polymerase,

carrying

incubation

clones from Fig. 3. Preferential

out as follows: BL21(DE3)(MuhPl)[E.

protein

Nuclear) X-Omat

for

fluor for 1 h. The fluor AR x-ray film at -80°C

standards

are indicated

on the

on the right.

E. coli genome (Fig. 3). Clones 1, 7, 9, 11, and 12 showed bands of 26 and 17.5 kDa, which have sizes predicted for the aroL and aroM products, respectively (DeFeyter et al., 1986). Clones 7, 11 and 12 also had a 46-kDa band corresponding to alkaline phosphatase (the phoA gene product). We could detect bands of approx. 27 and 50 kDa having sizes expected for the phoB and phoR gene products (Makino et al., 1986a,b), respectively, for clones 1 and 9 in overexposed gels (data not shown). It is interesting to note that the mini-Mu-coded products (MuA, MuB and Ner) are seen in reduced amounts in clones 1, 7, 9, 11, and 12, that contain the mini-Mu with the T7 promoter transcribing clockwise-oriented genes. This may be due to the presence of transcription terminating sequences downstream from the aroLM operon (DeFeyter et al., 1986). Four new genes were discovered in this region coding for products of approx. 37, 52, 66, and 95 kDa. The 37-kDa band was present in clones 7 and 12 but not 1, 11 or 9, indicating an ORF with a start codon located to the left of the leftmost Hind111 site (Fig. 3). Clones 7, 11, and 12 but not 9 or 1 produced a 66-kDa protein corresponding to a locus present between the leftmost Hind111 site and the

phoA gene (Fig. 3). The only known genes in this region are sbmA and bmQ (Bachmann,

1987; LaviAa et al., 1986). Neither the smbA gene product, which has an expected size of approx. 33 kDa based on transposon mutagenesis data (Lavifia et al., 1986), nor the brnQ gene product have been identified in SDS-PAGE gels. This suggests that the 66-kDa or the 37-kDa protein may be the product of bmQ or corresponds to new genes. Protein gels corresponding to plasmid clones 2, 3, 4, 5, 6, 8, and 10 showed a common 29-kDa band which has the size expected for the proC gene product (Deutch et al., 1982). Clones 6, 10 and also 5 (as indicated by longer exposures of the gel) shared a band of approx. 95 kDa. This would require a 3-kb DNA segment located between the sbcC gene, and the phoBR operon (as defined by clones 4 and 5; see Wanner and Chang, 1987). The transcriptional orientation of sbcC is unknown, but its size as determined by transposon mutagenesis is too small to account for a 95-kDa protein (Groisman, 1986). Clones 6 and 10 also share a 52-kDa protein band which is placed to the right of the phoBR operon (as defined by clones 5 and 6).

6

(c) Conclusions (1) Transposable elements have been used primarily to isolate mutants because their insertion usually results in a

ACKNOWLEDGEMENTS

null phenotype (Berg et al., 1989). Sometimes, however, the insertion of a transposon leads to gene activation, either by

assistance, W.F. Studier for strains, W. Barnes for providing the nt sequence of the phoA gene prior to publication,

transcription from a terminally located promoter in the case of IS2, IS3, Tn3 and TnlO, to cause constitutive expression

and H. Revel and R. Haselkorn for their interest and support. M.J.C. was supported by Public Health Service

of the downstream genes (Charlier et al., 1982; Ciampi et al., 1982; Heffron et al., 1979; Schwartz et al., 1988;

grant AI00468 from the National Institutes of Health. This work was supported in part by Public Health Service grant GM29067 from the National Institutes of Health.

Simons et al., 1983); or via the alteration region, as seen in the ISI- or I%-mediated

of the local DNA activation of the

E. coli bgl operon

(Reynolds et al., 1981). The mini-Mu replicon element Mud5294 described here (Fig. 1) has been used to make gene libraries and express cloned genes. It could also be used to mutagenize the chromosome, control

the expression of neighboring genes, generate conditional mutations or provide a convenient source of antisense RNA. Recently, Tn1721 (Ubben and Schmitt, 1987) Tn5 (Chow and Berg, 1988; Nag et al., 1988) and mini-Mu transposons (P. Olfson and M.J.C., unpublished results; E.A.G. and F. Heffron, unpublished results) have been engineering to carry regulatable bacterial or phage promoter sequences. (2) The mini-Mu replicon containing a T7 promoter should be useful to identify gene-product relationships and to establish the number of genes or translatable ORFs that exist in different bacterial genomes. This task, exemplified by the analysis of the region around the E. coliproC gene, could be accomplished by generating a DNA/SDS-PAGE protein band index of the organism with clones corresponding to 100 markers spaced around the chromosome. This would complement the restriction map (Kohara et al., 1987) the ordered cosmid library (Tabata et al., 1989) and the gene-protein index presently available. The latter identifies only 10% of the 2100 different polypeptides visualized in two-dimensional protein gels corresponding to cells grown under different conditions (Phillips et al., 1987). The generation of gene banks with the T7 promotercontaining mini-Mu in an E. coli strain that harbors the T7 RNA polymerase gene in the chromosome should allow the cloning of genes that are poorly expressed in heterologous organisms or that require the presence of an unlinked activator for proper expression. RNA can also be prepared for ‘chromosome walking’ or in vitro translation experiments. It may be possible to construct an analogous cloning system for animal cells using modified retroviruses or engineered mini-Mu transposons harboring replication origins and packaging signals of viruses with large genomes such as herpes simplex or Epstein-Barr virus to allow the transduction of larger DNA segments between cells.

We thank

B. Richard

and M. Pagratis

for technical

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