Gene, 104 (1991) 81-84

0 1991 Elsevier Science Publishers B.V. All rights reserved 0378-I 119/9l/SO3.50 81

GENE 05061

Construction of a novel suicide vector: selection for Escherichia coli HBlOl recombinants carrying the DNA insert

(Recombinant DNA; positive-selection cloning vehicle; suicide cloning vector; rcsB; colanic acid; capsular polysaccharide;

Klebsiella gene library)

Yoshichika Arakawa”, Rochaporn Wacharotayankun”, Michio Ohta”, Kazuaki Shoji b, Masanori Watahiki b, Toshinobu

Horii” and Nobuo Kato”

u Department of Bacteriology, Nagoya University School of Medicine, Nagoya 466 (Japan), and h Enzymological Research Laboratory, Nippon Gene Co., Ltd., Toyama 930 (Japan) Tel. (0764)51-6548

Received by R.E. Yasbin: 26 December 1990

Revised/Accepted: 10 April/2 May 1991 Received at publishers: 21 May 1991

SUMMARY

We constructed a new type of cloning vector, pERISH2, that transforms Escherichia colt’ HB 101 only when a foreign DNA

fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from

the chromosome of E. coli HBlOl and is involved in the regulation of colanic acid production. When E. coli HBlOl is

transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the

rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth

of E. coli HBlOl. Although E. coli HBlOl is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and

C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large

quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and

efficiently by the utilization of this new cloning vector.

INTRODUCTION

Yanisch-Perron et al. (1985) constructed a series of

unique cloning vectors, M 13mp phages and pUC plasmids,

which carry an N-terminal fragment of p-galactosidase-

encoding gene, IacZ’. When a foreign DNA insert is

ligated into the cloning site of the vector, the color of the

colonies or plaques turns from blue to white through

blockage of a-complementation by La&’ in the presence

of XGal and IPTG in the medium. Therefore, these vectors

are useful for distinguishing transformants harboring re-

combinant plasmid with a foreign DNA insert from those

harboring self-ligated vectors. This kind of vector has been

modified for utility, and many types of vectors are now in

Correspondence to: Dr. Y. Arakawa, Department of Bacteriology, Nagoya

University School ofMedicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466

(Japan) Tel. (052)741-2111, ext. 2053; Fax (052)731-9479

Abbreviations: bp, base pair(s); CFU, colony-forming unit(s); Cm,

chloramphenicol; cps, gene cluster for the biosynthesis of Klebsiella K2 capsular polysaccharide; d, deletion; IPTG, isopropyl-/?-o-thiogalactopy-

ranoside; kb, kilobase or 1000 bp; K., Klebsiella; LacZ, product ofgene

1ac.Z (/I-galactosidase); LacZ’, product of 3’-truncated gene IacZ’

(laczcc); LB, Luria-Bertani (medium); nt, nucleotide(s); rcsB and rcsC,

E. coli genes encoding RcsB and RcsC; RcsB, positive transcriptional

regulator for colanic acid synthesis; RcsC, putative environmental sensor

which activates RcsB post-translationally by phosphorylation; rmpA,

gene involved in the regulation of capsular polysaccharide biosynthesis,

isolated from a resident large plasmid of K. pneumoniae Chedid; Tc,

tetracycline; XGal, 5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside;

[ 1, denotes plasmid-carrier state.

82

use worldwide. Although several types of positive-selection

cloning vehicles have been constructed (Chang and

Modrich, 1983; Dean, 1981; Roberts et al., 1980; Vernet

et al., 1985), they are not necessarily in general use. We

propose rcsB as a new genetic marker of plasmid for the

construction of a suicide vector which enables one to

distinguish easily and exclusively transformants harboring

recombinant plasmid with a foreign DNA insert.

EXPERIMENTAL AND DISCUSSION

(a) Media and strains

Cells were grown in LB medium or LB-agar plate

(Maniatis et al., 1982) supplemented with appropriate

antibiotics. Bacterial strains used were E. coli HB 10 1 (F _,

hsdS20(r-,m-), recA13, ara-14, proA2, lucY1, galK2,

rpsL20(SmR), ~~1-5, m&l, supE44, k) (Maniatis et al.,

1982); JM109 (recA1, d(kzc-proAB), endA 1, gyrA96,

thi-1, hsdR17, reoll, supE44[F’ traD36, proA’B+

luclqZdM15]}; C600 (F-, thi-1, thr-1, leuB6, lucY1,

tonA21, supE44 A-); W3110 (a laboratory strain of E. coli

K-12); B (a wild-type strain of E. coli; Boyer and

Roulland-Dussoix, 1969); K. pneumoniae Chedid (a labora-

tory strain; Mizuta et al., 1983).

(b) Cloning of rcsB gene

Although HBlOl showed a mucoid phenotype with the

enhancement of colanic acid production when it was trans-

formed by rmpA (Nassif et al., 1989). JM109 did not. We

speculated that the gene(s) in JM109 might not suffice for

the biosynthesis of colanic acid. Therefore, we cloned a

gene relevant to the mucoid phenotype from HBlOl into

JM109 as follows. EcoRI-digested genomic DNA frag-

ments of HBlOl were ligated into a plasmid vector

pACYC 184 for the construction of a gene library of HB 10 1.

E. coli JM109 harboring pROJ3SE, which carries rmpA,

was transformed by this gene library, and colonies pro-

ducing a large amount of colanic acid were isolated on an

LB agar plate supplemented with 15 pg Tc/ml. A recom-

binant plasmid, pCPSX1, was identified in the transfor-

mant producing a large amount of colanic acid.

(c) Characterization of pCPSX1

Although JM109 could be transformed by pCPSX1, no

E. coZi HBlOl[pCPSXl] transformant was isolated. The

pCPSX1 was prepared from JM109[pCPSXl] in a large

quantity. The restriction map of pCPSX1 was determined

and its functional region was identified by deletion-kit

(Nippon Gene Co., Ltd., Japan) (Fig. 1). Sequencing of the

functional region was achieved by the dideoxy method of

Sanger et al. (1977) and we found an nt sequence highly

homologous with the rcsB (Stout and Gottesman, 1990).

p K

pCPSX1

Fig. 1. Restriction maps of pCPSX1 and pERISH2. Arrows within the

plasmid indicate the direction of transcription and approximate extent of

gene rcsB. Open portion of arrow indicates the promoter region of WE.

The S-kb Hind111 fragment on pCPSX1 was subcloned into pHSG398H,

and the resulting plasmid was designated pERISH2. The thick lines of

each plasmid indicate the region of the vector plasmid. Open boxes with

CmR or TcR specify the positions of the Cm- or Tc-resistance-encoding

genes. The site of the DNA replication origin of each plasmid is indicated

as on‘ (0). E, EcoR1; H, HindHI; K, @I; P. PstI.

(d) Construction of suicide vector pERISH2

The EcoRI-SphI fragment in the polylinker site of

pHSG398 (Takeshita et al., 1987) was removed using a

blunting kit (Takara Co., Ltd., Japan) and the resulting

plasmid was designated pHSG398H. The 5-kb Hind111

fragment of pCPSX1 was subcloned into pHSG398H and

a recombinant plasmid, pERISH2, was constructed

(Fig. 1). Transformation of HBlOl by pERISH2 was

blocked as was observed in pCPSX1.

(e) Southern hybridization analysis

E. coli HB 10 1 is a hybrid strain of E. coli K- 12 and E. co/i

B. Therefore, we attempted to determine the strain from

which rcsB was derived. The PstI-KpnI fragment of

pCPSX1 carrying a promoter and a part of rcsB was used

as a DNA probe to detect the nt sequence homologous with

the cloned rcsB gene among several strains of E. coli, E. coli

83

123456

kb 23.1 *

9.4 *

6.6 *

4.4 *

2.3 * 2.0 *

Fig. 2. Southern hybridization. EcoRI-digested chromosomal DNAs

were blotted onto a nylon membrane after agarose gel (1%) electro-

phoresis. PsrI-KpnI fragments of pCPSXl (Fig. 1) carrying a part of gene

rcsB were used as the “P-labeled probe. Labeling of the DNA probe and

hybridization were done according to Maniatis et al. (1982). Arrowheads

on the right margin indicate the strong hybridization between the probe

and chromosomal DNAs prepared from HBlOl, JM109, C600 and

W3110 but only faint hybridization with the chromosome ofE. coli B was

observed. Lanes: 1, HindHI-digested phage 1 DNA (only sizes on left

margin); 2,E. co/i B; 3, HBIOI; 4, JM109; 5, C600; 6, W3110.

B, HBlOl, JM109, C600, and W3110. Chromosomal

DNAs were prepared from these strains, digested by EcoRI,

and blotted onto a nylon membrane after electrophoresis

according to conventional methods (Maniatis et al., 1982).

The DNA probe hybridized strongly with the chromosomal

DNA of HB 101, JM109, C600, and W3 100, but only faintly

with that of E. coli B (Fig. 2). The length of the EcoRI-

digested chromosomal DNA fragments with which the

probes hybridized was apparently the same in HBlOl,

JM109 and C600, while that of W3 110 was slightly longer.

From these results, we concluded that rcsB of HBlOl

derived from E. coli K-12.

(f) Construction of a Klebsiella pneumoniae gene library

Chromosomal DNA of K.pneumoniae Chedid was

digested partially with PstI and the DNA fragments longer

than 20 kb were purified from agarose NA (Pharmacia)

after electrophoresis. The purified DNA fragments (10 pg)

were ligated into the PstI site in the functional region of

pERISH2 (30 pg) (Fig. 3). Competent cells (1 x 10’ CFU)

of E. co/i HB 101 suspended in 200 ,ul of conventional trans-

chromosome of K.pnemoniae Chedid

@

1

PstI digestion

(partial) --- --- -- ---

p=oC;; of long DNA fragments

&

pERISH2

P

0

PstI digestion

plasmids with ligation

transformation

+ t grow die

Fig. 3. Construction of a gene library ofK. pneumoniae Chedid. Chromo-

somal DNA of K. pneumoniue Chedid was digested partially by PsrI.

DNA fragments longer than 20 kb were purified and ligated to PstI-

digested pERISH2. We did not treat PstI-digested pERISH2 with

alkaline phosphatase because we selected against the self-ligated vectors.

Competent cells of E. coli HBlOl prepared by conventional methods

were transformed by these recombinant plasmids’ mixture containing

some amounts of self-ligated vector pERISH2. Although the cells which

receive self-ligated vector plasmid carrying the intact gene rcsB cannot

grow on LB-agar plate supplemented with 35 pg Cm/ml, those harboring

recombinant plasmid carrying a foreign DNA insert in rcsB can. Coiled

and wavy lines represent chromosomal DNA. Letter P under pERISH2

or beside recombinant plasmids indicate PslI restriction site.

formation buffer (50 mM CaCl,/lO mM Tris * HCl pH 7.5)

were transformed by using these recombinants (half of the

ligated DNA). After heat shock, transformed cells were

incubated with 2 ml of high-competent broth (Nippon Gene

Co., Ltd.) for 30 min at 37’ C. Transformed cell suspension

(0.2 ml) was inoculated and cultured on each LB-agar plate

supplemented with 35 pg Cm/ml. Colonies were isolated on

LB-agar plate after 18 h incubation at 37 ‘C. Under these

experimental conditions, we obtained 0.5-2 x lo3 colonies

on each LB-agar plate. 100 transformants were picked up

at random and cultured for the preparation of recombinant

plasmid. Plasmid DNA was prepared from each culture

84

and its size was tested by agarose-gel electrophoresis.

Recombinant plasmid with a long DNA insert was

harbored by 98% of tested transformants.

(g) Cause of lethality

(1) Two regulatory pathways for the biosynthesis of

colanic acid have been found (Gottesman et al., 1985). It is

speculated that one of them may belong to a two-

component regulatory system, RcsC/RcsB, (Stout and

Gottesman, 1990). In this system, the positive regulator

RcsB may be activated by RcsC, a transmembrane protein

and an environmental sensor, through phosphorylation. It

is speculated that activated rcsB enhances the transcription

of mRNA from cpsA-E cluster for the synthesis of colanic

acid in E. coli. Many kinds of two-component regulatory

systems have been found (Stock et al., 1989). All of these

two-component regulatory systems, such as EnvZ-OmpR,

play an important role in bacterial growth (Aiba et al., 1989;

Miller et al., 1989). Some cross-talk in the transfer of the

phosphoryl group has been observed among these systems

(Stock et al., 1989). Therefore, if the amount of intracellular

RcsB is excessively increased, homeostasis in the bacterial

cell may be destroyed by the disturbance of transcriptional

regulations through the cross-talk of these two-component

regulatory systems, possibly resulting in the death of bacte-

rial cells.

(2) Although neither E. coli B nor HBlOl were trans-

formed by pCPSX1 or pERISH2, only HB 101 had the rcsB

gene on the chromosome as was shown by Southern

hybridization. All the strains of E. coli K-12 tested had the

rcsB gene and they were transformed by pERISH2. These

results suggest that presence or absence of the rcsB gene on

the chromosome may not be critical in the inhibition of

bacterial growth by the transformation with exogenous

rcsB. E. coli HB 101 is a hybrid strain of E. coli B and K-12

and we found that rcsB of HB 10 1 derived from K- 12. E. coli B, which has no gene homologous with rcsB of HB 101, was

not transformed by pERISH2 as was observed in HB 10 1.

An RpoN-like promoter was found upstream from rcsB (Stout and Gottesman, 1990). It is therefore suggested that

the lethal response to exogenous excessive rcsB in HBlOl

may derive from E. coli B, and that the transcriptional

regulation of rcsB may differ between HBlOl and K-12. It

was reported that multicopy expression of rcsB is lethal in

rcsC mutants which carry cps-fuc fusion (Julie et al., 1988).

However, HBlOl carries intact rcsC and cps because it

produces a large amount of colanic acid when transformed

by rmpA (Nassif et al., 1989). Further analysis on the

mechanism of lethality should be done.

(h) Conclusions

(1) Use of rcsB as a new genetic marker of the suicide

cloning vector, pERISH2, makes it possible to establish a

simplified method for the positive selection of transfor-

mants harboring arecombinant plasmid with a DNA insert.

(2) A gene library of chromosomal DNA of

K. pneumoniue is constructed easily and efficiently by use of

the newly developed suicide vector, pERISH2.

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