Plasmid 52 (2004) 57–62

www.elsevier.com/locate/yplas

Integrative plasmid vector for constructingsingle-copy reporter systems to study gene regulation

in Rhizobium meliloti and related species

S. Ferenczi,a A. Ganyu,a B. Blaha,a,b S. Semsey,a,1 T. Nagy,a,2

Z. Csiszovszki,a,b L. Orosz,a,b and P.P. Pappa,*

a Institute of Genetics, Agricultural Biotechnology Center, G€od€oll, Szent-Gy€orgyi A. 4. H-2100, Hungaryb Department of Genetics, Faculty of Science, L�or�and E€otv€os University, Budapest, P�azm�any P. 1/C., H-1117, Hungary

Received 11 March 2004, revised 18 April 2004

Available online 20 May 2004

Abstract

The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species

belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera. It might also function in many

other bacterial species provided that both the target site (attB) and the required host factor(s) are present. Here we report

on the construction of a new integrative vector that can be utilized in gene regulation studies. It provides an opportunity

to create a single-copy set-up for characterizing DNA–protein interactions in vivo, in a wide range of bacteria. To

demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to

wild type operators was studied. The assay system provided highly reproducible quantitative data on repression.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Rhizobium meliloti; Bacteriophage; Site-specific recombination; DNA–protein interactions; Reporter system; Repressor

1. Introduction

Genetic studies and biotechnological applica-

tions frequently require the insertion of DNA

* Corresponding author. Fax: +36-28-526-145.

E-mail address: ppapp@abc.hu (P.P. Papp).1 Present address: Laboratory of Molecular Biology, Na-

tional Cancer Institute, NIH, Bethesda, MD 20892-4264, USA.2 Present address: University of Newcastle Upon Tyne

School of Cell and Molecular Biosciences Newcastle, Upon

Tyne NE1 7RU, UK.

0147-619X/$ - see front matter � 2004 Elsevier Inc. All rights reserv

doi:10.1016/j.plasmid.2004.04.005

fragments, carrying different kinds of genetic in-

formation, into the chromosome. Specific and

stable engineering of the chromosome may be

difficult in most of the bacterial species due to the

lack of suitable and efficient tools. In gene regu-

lation studies, this problem can be circumvented

by performing the tests in a model organism, such

as Escherichia coli, where genetic changes canbe made relatively easily. Since the intracellular

environment (e.g., ionic composition, pH value,

availability of host factors, and relations to other

ed.

58 S. Ferenczi et al. / Plasmid 52 (2004) 57–62

controlled processes) may influence or bias theoutcome, it is more accurate to study in vivo gene

regulation in the original host.

High fidelity in vivo studies on gene regulation

requires a single-copy system. This practically

means that the measuring unit, comprising the

reporter gene preceded by the control site and a

cognate promoter, is built into the chromosome of

the host cell. The source of the trans-acting regu-lator protein can be either the chromosome or a

multi-copy plasmid. Site-specific recombination is

an efficient way for targeted integration of the unit

to be assayed into the chromosome.

The temperate phage 16-3 of Rhizobium meliloti

41 has been studied thoroughly. Two of the major

phage functions have been localized to the middle

section of the phage chromosome (GenBank Ac-cession Nos. AJ131679 and AJ519534). One of the

major functions is the immunity function found to

be essential for lysogenic development. Its main

structural elements, the c and immX repressor

genes, cognate cis sites, and the interactions be-

tween the C repressor and its operator sites have

been investigated in detail (Csiszovszki et al., 2003;

Dallmann et al., 1987, 1991; Dudas and Orosz,1980; Orosz, 1980; Orosz et al., 1980; Papp et al.,

2002). OL and OR type operators were found to be

structurally different (50-ACAA-4 bp-TTGT-30 and

50-ACAA-6 bp-TTGT-30, respectively) (Papp et al.,

2002). The other phage function in the central

chromosome segment ensures the integration of

the phage genome into the chromosome or exci-

sion of the prophage from the chromosome. Thesite-specific recombination system of phage 16-3

and its key elements (int, xis, and attP) have

also been identified and characterized in detail

(Dorgai et al., 1993; Olasz et al., 1985; Semsey

et al., 1999). The target sequence, attB of the

bacterial chromosome, was located within a pro-

line tRNA(CGG) gene (Papp et al., 1993a). It was

demonstrated that a plasmid, containing the 16-3

integrative elements, was able to integrate into the

chromosomes of various bacteria of biotechno-

logical and agricultural importance. The target

sites, as determined in species belonging to the

Rhizobium, Bradyrhizobium, Azorhizobium, and

Agrobacterium genera, were all within the same

proline tRNA(CGG) genes (Semsey et al., 2002).

In this paper, we report the construction of anintegrative vector suitable to develop a single-copy

reporter system for characterizing DNA–protein

interactions in vivo. The use of the system was

demonstrated in a comparative study of the op-

erator–repressor interactions of phage 16-3 in R.

meliloti 41 (i.e., in the natural host of the phage)

and also in Agrobacterium tumefaciens.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Escherichia coli strain DH5a (Hanahan, 1983)

was used in all cloning experiments and served as

a host of donor plasmids used to conjugate intothe recipient bacterial strains such as R. meliloti

41 and A. tumefaciens GV2260. The triparental

mating method was used to transfer the different

plasmids resident in E. coli into the recipient

bacteria. E. coli harboring pRK2013 (Figurski

and Helinski, 1979) or pCU101 (Thatte et al.,

1985) served as a helper for plasmids with RP4

mob or pCU1 mob, respectively. E. coli wasgrown in Luria broth at 37 �C, R. meliloti and A.

tumefaciens were grown in yeast–tryptone broth

(YTB) (1% tryptone, 0.1% yeast extract, 0.5%

sodium chloride, 1mM magnesium chloride, and

1mM calcium chloride, pH 7.0) at 28 �C. For

plating bacteria, both Luria broth and YTB were

supplemented with 1.5% agar, resulting in Luria

agar and YTA, respectively. Conditions for tri-parental matings are described in Semsey et al.

(2002).

2.2. DNA procedures

Basic DNA manipulations and molecular

techniques were employed as described in Sam-

brook et al. (1989). Extraction of DNA fromagarose gels was done with QIAEX II Gel

Extraction Kit (QIAGEN). PCR conditions, used

for detecting site-specific integration of different

plasmids into the chromosome of R. meliloti and

A. tumefaciens, are described in Semsey et al.

(1999). Nucleotide sequence determination was

performed by the dideoxy chain termination

S. Ferenczi et al. / Plasmid 52 (2004) 57–62 59

method (Sanger et al., 1977) with the fmol DNAcycle sequencing system (Promega).

2.3. Construction of plasmids

To build pSEM211, the 4.2 kb DraI fragment of

pMLB1109 (Papp et al., 1993b), containing a

promoterless lacZ gene preceded by four T1T2

terminators in a tandem arrangement, was inserted

into pSEM91 at the filled in Acc65I site. Plasmid

pGSB1 was created using several steps. pSEM155

was constructed by inserting a 261 bp long frag-

ment, containing the functional attP region ofphage 16-3, into pSEM143 (Semsey et al., 2002).

The expression panel of pSEM164 (Semsey et al.,

1999), producing the integrase protein of phage 16-

3, inserted into pSEM155 resulted in the plasmid

pSEM289. The kanamycin resistance gene of

pSEM289 was replaced with a spectinomycin

cassette obtained from pCU996X (Banerjee et al.,

1992) and insertion of the promoterless lacZ genefrom pMLB1109 resulted in the plasmid pGSB1.

pGSB42 and pGSB62 were constructed by inser-

tion of synthetic oligonucleotides into the KpnI site

of pGSB1. The sequences of the oligonucleotides

between the KpnI sites are shown in Table 1 for

each plasmid. All plasmids used in this study are

listed in Table 1, including plasmid pPM232 (Papp

et al., 2002) used for donating the repressor.

Table 1

Plasmids used in the studies

Plasmid Relevant features

pRK2013 Helper in conjugative transfer of plasmid with R

pCU101 Helper in conjugative transfer of plasmid with pC

pCU996X Source of spectinomycin cassettes (X)pMLB1109 Source of promoterless lacZ gene

pSEM143 pSC101 replicon, pCU1 mob, KmR

pSEM164 Source of expression panel to produce 16-3 Int p

pSEM91 Expression vector, pCU1 replicon, RP4 mob, Km

pPM232 Wild type c gene of 16-3 cloned into pSEM91

pSEM155 pSC101 replicon, pCU1 mob, attP, KmR

pSEM289 pSC101 replicon, pCU1 mob, attP, and int gene

pSEM211 pCU1 replicon, RP4 mob, KmR, promoterless lac

pGSB1 pSC101 replicon, pCU1 mob, attP, and int gene

pGSB42 Contains OL2 operator within the promoter in pG

50-TTGACTCTACAATTGATTGTATATAGT -3

pGSB62 Contains OR2 operator within the promoter in pG

50-TTGACTACAATTGTAGTTGTATATAGT -3

2.4. Calculation of repression levels (R values)

b-Galactosidase assays and calculations of

promoter activities (expressed in Miller Units

(MU)) were carried out as described in Miller

(1972). Repression values (R) were calculated

using the following equation:

R ¼ 1� promoter activity in the cell when repressor was added from plasmid

promoter activity in the cell without repressor:

3. Results

3.1. Construction of the basic integrative vector

A new integrative plasmid vector, pGSB1, has

been constructed carrying the site-specific recom-

bination system of phage 16-3 (Fig. 1). It uses the

replicon of pSC101, an E. coli specific plasmid. Italso has mob region of pCU1 origin that facilitates

conjugative transfer in a broad range of bacteria by

using the pCU101 helper plasmid, the int gene and

attP region of phage 16-3 that ensure efficient in-

tegration into the chromosome in several bacteria,

the lacZ gene that serves as a reporter gene and the

spectinomycin resistance gene that allows direct

selection. The region preceding the promoterlesslacZ gene contains a unique KpnI/Acc65I restric-

tion site for cloning fragments containing a pro-

moter and a control site in a desired arrangement.

Reference

P4 mob Figurski and Helinski (1979)

U1 mob Thatte et al. (1985)

Banerjee et al. (1992)

Papp et al. (1993b)

Semsey et al. (2002)

rotein Semsey et al. (1999)R Semsey et al. (1999)

Papp et al. (2002)

This work

of phage 16-3, KmR This work

Z This work

of phage 16-3, lacZ, SpR This work

SB1 This work0

SB1 This work0

Fig. 1. The major components of plasmid pGSB1. KpnI/Acc65I

site is unique and suitable for cloning. Positive clones carrying

the desired promoter/control-site unit in the appropriate ori-

entation can be selected by color in the presence of X-gal.

60 S. Ferenczi et al. / Plasmid 52 (2004) 57–62

The T1T2 terminators restrains upstream tran-

scription from entering into the lacZ gene.

3.2. Construction of different promoter/operator

units for characterizing 16-3 repressor–operator

interactions

To investigate 16-3 repressor binding in vivo, an

artificial promoter/operator unit was constructed

by oligonucleotide synthesis. In planning the se-

quence of the promoter/operator unit, the sequences

of the PR, PL, and PC promoter regions of phage 16-3 were taken into consideration (Dallmann et al.,

1987; Elo et al., 1998). The 14-bp long OR2 operator

sequence (Papp et al., 2002) was inserted between

the)35 and the)10 elements of ar70 type promoter

by keeping the spacing 15 bp long. To achieve har-

mony between the sequences of the)10 element and

the spacer, incorporation of any nucleotide was al-

lowed in two positions within the )10 elementduring synthesis of the oligonucleotide (50-TTG

ACTACAATTGTAGTTGTATNTANT-30; bold

letters indicate the conserved nucleotides of the

operator, the italicized letters promoter elements,

and N undefined bases). Oligonucleotides repre-senting the two strands were annealed and cloned

into pSEM211. Colonies following transformation

of E. coli were collected and the plasmids were in-

troduced intoR. meliloti by conjugation. Sequences

of the promoter/operator units were determined in

some recombinant plasmids derived from blue col-

onies. A promoter with TATAGT for the )10 ele-

ment was selected and served as a basis to createfurther promoter overlapping the 12-bp long OL2

(Papp et al., 2002) operator. This promoter/opera-

tor variant has been constructed also from oligo-

nucleotides by maintaining the )35 and )10elements of the active promoter unchanged and by

trying to minimize the differences in the spacer

region (sequences are shown in Table 1). The pro-

moter/operator unit was cloned into pGSB1 andthe resulting plasmids, pGSB42, was introduced

into R. meliloti by conjugation.

Since neither pGSB1 nor its derivatives were

able to replicate in R. meliloti, spectinomycin was

included in the medium to select for colonies

containing the plasmid integrated into the host

genome. The presence of X-gal allowed the de-

tection of promoter activity. In the case of bluecolonies, b-galactosidase activity was measured

and the specific integration of the plasmid was

confirmed by PCR using att-specific primers.

3.3. Determination of repression (R) in vivo

Plasmids containing the different promoter/op-

erator units (pGSB42 and pGSB62) were inte-grated into the genome of R. meliloti and A.

tumefaciens, and single colonies were purified. To

determine the repression on the different opera-

tors, pPM232 or pSEM91 were introduced into

these cells by conjugation. The plasmids supplied

wild type repressor or served as repressorless

control, respectively. b-Galactosidase activities

were then determined and repression values (R)were calculated. The results are shown in Table 2.

4. Discussion

We have developed a new and efficient inte-

grative plasmid vector and demonstrated that it is

Table 2

Promoter activities and repression values in R. meliloti and A. tumefaciens

Repressor None Wild type C

Operator R. meliloti A. tumefaciens R. meliloti A. tumefaciens

OL2 MU 46� 1 220� 5 10� 0 45� 1

R — — 0.78 0.80

OR2 MU 60� 3 241� 2 11� 1 40� 1

R — — 0.81 0.83

MU, b-galactosidase activity in Miller Unit; R, repression (see calculation in Section 2); and nt, not tested.

S. Ferenczi et al. / Plasmid 52 (2004) 57–62 61

suitable for making recombinant R. meliloti and A.

tumefaciens strains carrying the transgene in a

single copy, integrated into the host chromosome.

To construct this plasmid we utilized the integra-

tive recombination system of R. meliloti phage 16-3. We also demonstrated the usefulness of the

plasmid in quantitative assays of gene regulation

using the repressor/operator–promoter system of

phage 16-3.

Our data showed that the sequence differences

in the different promoter/operator units did not

result in huge variations in promoter activities

(14MU in R. meliloti and 21MU in A. tumefac-

iens). We have noticed that the activities of the

promoters were higher in A. tumefaciens than in

R. meliloti (compare data in the first two columns

of Table 2).

Our results are consistent with two previous

observations: (i) In the single copy set-ups, similar

repression values were obtained in E. coli, where

the integrative system of phage k was used, and inR. meliloti and A. tumefaciens, where the 16-3 in-

tegrative system was used (Papp et al., 2002 and

Table 2). (ii) Binding of the C repressor protein to

the wild type operators, OL2 and OR2, resulted in

efficient repression despite the differences in their

structure.

From our present and previous studies we

conclude that binding of the 16-3 repressor todifferent operators was not influenced by the

organism used in the assays. The very similar

repression values measured in R. meliloti and A.

tumefaciens show that our plasmid system is suit-

able to establish single copy set-ups in the natural

host of phage 16-3, from which the site-specific

recombination system was derived, as well as in

A. tumefaciens. We assume that plasmid pGSB1can be used for gene regulation studies in all the

species where the 16-3 integrative system has been

successfully used, and it may also function in their

related species.

Acknowledgments

We thank Korn�elia Sz�or�ath G�al, Magdolna

T�oth P�eli, and Csilla S�anta T€or€ok for excellent

technical assistance and Andrei Trostel for dis-

cussion and helpful comments on the manuscript.

This work was supported by grants from the

Hungarian Scientific Research Fund (OTKA) (T023695, T 032205, and T 032255), the National

Research and Development Program (NKFP)

(OM 0028/2001 and OM 278/2001) and the Hun-

garian Academy of Sciences (MTA/TKI/AKT-F

1999–2001 and MTA/TKI/AKT-F 2003–2006).

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