Molecular Microbiology (2001) 42(1), 195–204
RepA negatively autoregulates the transcription of the
repABC operon of the Rhizobium etli symbiotic plasmid
basic replicon
Miguel A. Ramı́rez-Romero,1 Juan Téllez-Sosa,1
Humberto Barrios,2 Angeles Pérez-Oseguera,1
Vania Rosas1 and Miguel A. Cevallos1*
1
Programa de Evolución Molecular, Centro de
Investigación sobre Fijación de Nitrógeno, Universidad
Nacional Autónoma de México, Apartado Postal 565-A,
Cuernavaca, Morelos, México.
2
Departamento de Biologı́a Estructural, Instituto de
Biotecnologı́a, Universidad Nacional Autónoma de
México, Apartado Postal 510–3, Cuernavaca, Morelos,
México.
Summary
The basic replicon of Rhizobium etli CE3, like other
members of the repABC plasmid family, is constituted
by the repABC operon. RepC is essential for
replication, and RepA and RepB play a role in plasmid
segregation. It has been shown that deletion deriva-
tives lacking the repAB genes have an increased copy
number, indicating that these genes participate in
the control of plasmid copy number. RepA is also
a trans-incompatibility factor. To understand the
regulation of the repABC operon, in this paper: (i)
the transcription start site of the repABC operon was
determined; (ii) the promoter region was identified by
site-directed mutagenesis of the putative 235 and
210 hexameric elements; and (iii) RepA was recog-
nized as a negative regulator of the transcription of
the repABC operon.
Introduction
Rhizobium etli CE3 is a soil bacterium with the ability to
recognize and induce nitrogen-fixing nodules on bean
plants. This strain has six plasmids of high molecular
weight and low copy number; one of them, the symbiotic
plasmid (p42d), harbours most of the genes required for
nodulation (nod genes) and nitrogen fixation (nif and
fix genes). The elements that this plasmid requires for
autonomous replication, segregation and plasmid copy
number regulation reside in a HindIII fragment of 5.6 kb.
Accepted 18 July, 2001. *For correspondence. E-mail mac@
cifn.unam.mx; Tel. (152) (7) 311 46 63; Fax (152) (7) 317 74 80.
Q 2001 Blackwell Science Ltd
Sequence analysis of this fragment showed the presence
of the repA, repB and repC genes and the conserved
intergenic region between repB and C, which character-
izes the members of the repABC plasmid family (Ramı́rez-
Romero et al., 1997).
The repABC plasmids lack some characteristics that
are commonly found in other large plasmids of low
copy number. For example, they lack iterons, which are
repetitive sequences that participate in regulating tran-
scription and plasmid copy number in other unicopy
plasmids such as plasmids F or P1.
Population studies indicate that repABC plasmids are
commonly found within the Rhizobiaceae family (Turner
et al., 1996; Rigottier-Gois et al., 1998), and the complete
sequence of the replicator regions of several repABC
plasmids from this bacterial family has been determined (see
Table 1). These plasmids are not exclusive to the
Rhizobiaceae, as they have been identified recently
in Paracoccus versutus and Rhodobacter capsulatus, two
a-proteobacteria, indicating that these plasmids are pro-
miscuous (Bartosik et al., 1998; Turner and Young, 2001).
The repABC plasmids encompass several incompat-
ibility groups. The so-called nopaline-type (i.e. pTiC58)
and octopine-type (i.e. pTiB6S3) Ti plasmids belong to the
same incompatibility group, but not to the incompatibility
group of the root-inducing plasmid pRiA4b (Hooykaas and
Schilperoort, 1984). Plasmid pTiC58 is compatible with the
symbiotic plasmid p42d of R. etli CE3, indicating that they
belong to distinct incompatibility groups (Brom et al.,
2000). Further evidence that the repABC family embraces
several incompatibility groups is provided by hybridization
studies, which demonstrate that more than one repABC
plasmid can co-exist in the same strain (Rigottier-Gois
et al., 1998).
A comparative sequence analysis of the repABC genes
showed that RepA and RepB have some similarity with
partition proteins from several prokaryotic chromosomes
and with partition proteins from plasmids P1 and F
( par/sop ) (Turner and Young, 1995; Ramı́rez-Romero
et al., 1997; Møller-Jensen et al., 2000). In contrast, RepC
is found in members of two closely related plasmid
families: the repABC family and the repC family. Genetic
analysis of the replicator region of plasmid p42d showed
that RepC is indispensable for replication (the initiator
protein) and that RepA and RepB products participate in
196 M. A. Ramı́rez-Romero
plasmid stability. The same analysis demonstrated that
the repA, B and C genes are organized in a single operon.
Deletion derivatives lacking the repA and B genes are
unstable, but they have an increased copy number,
indicating that RepA and RepB participate not only in
segregation but also in plasmid copy number control.
RepA is also a trans-acting incompatibility factor
(Ramı́rez-Romero et al., 2000).
Some elements that participate in replication, partition-
ing, plasmid copy number control and incompatibility
have been described; however, little is known about the
regulatory properties of the repABC operons.
With this aim, some elements required in the transcrip-
tional regulation of the repABC operon are described
here. First, the promoter and the transcriptional start site of
the repABC operon were identified and, secondly, RepA
was recognized as an essential element in the negative
regulation of the transcription of the repABC operon.
Results
Expression of repA::gusA fusions containing different
length upstream regions of repA
Recently, it was shown that a DNA segment starting
160 bp upstream of repA and ending 500 bp downstream
of repC contained all the elements required for stable
replication in an R. etli strain cured of its symbiotic plasmid.
It was also determined that the repABC genes are
organized in a single transcriptional unit, indicating that the
repABC promoter and its associated regulatory sequences
reside within the 160 bp upstream of repA (Ramı́rez-
Romero et al., 2000). The b-glucuronidase activity of a
repA::gusA transcriptional fusion containing the 160 bp
upstream of repA (pGUS-PWT160) can easily be detected
when it is introduced into a pSym-cured derivative of R. etli.
A similar construction containing 82 bp upstream of repA
(pGUS-PWT82) retained the same ability to express b-
glucuronidase activity in a R. etli pSym-cured derivative,
but a construction with only 77 bp upstream of repA
(pGUS-PWT77) did not (Fig. 1A). These results indicate
that essential transcription elements are encoded within
the region between 77 bp to 82 bp upstream of the initiation
codon of repA.
When these repA::gusA fusions were transformed
into Escherichia coli BW21038, none of them was able
to express the reporter gene, indicating that the R. etli
promoter located in this region was not recognized by the
transcription machinery of E. coli.
Transcription start site of the repABC operon
To determine the transcription start site of the repABC
operon, a primer extension analysis was performed in
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204
strain CFNX107 (pGUS-PWT160). The signal was
obtained in the G nucleotide located 45 bp upstream of
the initial repA codon (Fig. 2). Sequences containing the
E. coli s70 promoter consensus [TTGACA(N16218)TATA
C/A A/T] were not found. However, a sequence with
weaker similarity to that consensus [TTGCTC(N16)
TCTAAT] was situated 7 bp upstream of the transcription
start site (see Fig. 1A) (Lisser and Margalit, 1993;
Ramı́rez-Romero et al., 1997).
Mutational analysis of the repABC promoter region
To demonstrate that the proposed sequence encodes a
functional promoter, five mutant derivatives were con-
structed from the repA::gusA fusion carried on plasmid
pGUS-PWT95. First, the putative 235 element was
altered, changing sequence (TTGCTC) to (TGTTTC) to
generate construct pGUS-PMB35. Secondly, the putative
210 element was changed from (TCTAAT) to (TCGCCT)
to produce construct pGUS-PMB10. Thirdly, the putative
235 element (TTGCTC) was changed to (TTGACA) to
match the consensus E. coli 235 element, and this
construct was named pGUS-PCS35Ec. Fourthly, the
putative 235 and 210 elements were changed at the
same time to match the E. coli consensus to generate
plasmid pGUS-PCS3510Ec. Fifthly, plasmid pGUS-PEM
was obtained by changing the spacer sequence located
between the 235 and 210 elements from (AAAATGCA
GAATCGGC) to (TGCATGCAGAATGAGA). These con-
structs were introduced into R. etli CFNX107 and E. coli
gusA – strain BW21038, and their b-glucuronidase
activities were determined. A summary of the results is
shown in Fig. 1C.
Constructs pGUS-PMB35 and pGUS-PMB10 did not
express b-glucuronidase activity in either R. etli or E. coli.
In contrast, the b-glucuronidase activity detected in an
R. etli strain carrying pGUS-PEM was not affected. The
finding that changes introduced in the putative 210 or 235
elements, but not those located in the spacer region
between these two elements, interfere drastically with
transcription indicate that all structural elements of the
promoter were identified correctly.
The construct carrying a promoter matching the E. coli
consensus (pGUS-PCS3510Ec) was able to express
b-glucuronidase activity in both R. etli CFNX107 and
E. coli (Fig. 1C and D) The b-glucuronidase activity of
this construct in R. etli CFNX107 was lower than that
obtained with the wild-type promoter in the same genetic
background (Fig. 1C).
RepA negatively regulates its own transcription
As described above, the b-glucuronidase activity of a
repA::gusA transcriptional fusion containing the 160 bp
 RepA is the negative regulator of the repABC operon 197

Table 1. The repABC plasmids of the Rhizobiaceae family whose replicator regions have been sequenced.

Species Plasmid Relevant characteristic Reference

Agrobacterium tumefaciens pTiC58 Tumour-inducing plasmid Li and Farrand (2000)

pTiB6S3 Tumour-inducing plasmid Tabata et al. (1989)
pTi-SAKURA Tumour-inducing plasmid Suzuki et al. (1998)
pAtC58 Cryptic plasmid AF283811a
Agrobacterium rhizogenes pRiA4b Root-inducing plasmid Nishiguchi et al. (1987)
Rhizobium etli p42d Symbiotic plasmid Ramı́rez-Romero et al. (1997)
Rhizobium leguminosarum pRL8JI Cryptic plasmid Turner and Young (1995)
Rhizobium sp. pNGR234a Symbiotic plasmid Freiberg et al. (1997)
Mesorhizobium loti pMLa Cryptic plasmid Kaneko et al. (2000)
pMLb Cryptic plasmid Kaneko et al. (2000)
Sinorhizobium meliloti pExo Plasmid carrying Chain et al. (2000)
exopolysaccharide genes

a. GenBank accession number.

upstream of repA (pGUS-PWT160) can be detected when
it is introduced into an R. etli pSym-cured derivative
(CFNX107). However, when the same construct is
transferred into a strain with the symbiotic plasmid
(CFNX101), its capacity to express b-glucuronidase is
severely diminished, suggesting that some pSym-encoded
factor, most probably related to its replication functions,
represses the expression of the repA::gusA fusion (Fig. 3).
To identify those elements that participate in the
negative regulation of the repABC operon, six different

Fig. 1. A. DNA alignment of the upstream regions of the repA::gusA fusions. Numbers in brackets indicate the number of nucleotides upstream of the
initial codon of repA contained in each repA::gusA fusion. The initial repA codon is in italic. The G nucleotide marked in bold face corresponds to the
transcription start site. The promoter 210 and 235 elements are indicated in white boxes. The broken line indicates the deleted DNA. Black boxes
indicate the introduced mutations. Arrows show the position of an inverted repeat element. Asterisks indicate the G nucleotides protected by RepA in
the genomic DMS footprinting assay.
B. Delimitation of the promoter region. b-Glucuronidase activities of R. etli CFNX107 transconjugants containing repA::gusA fusions with upstream
regions of different lengths: bar 1, CFNX107 (pBBMCS53); bar 2, CFNX107 (pGUS-PWT160); bar 3, CFNX107 (pGUS-PWT95); bar 4, CFNX107
(pGUS-PWT82); bar 5, CFNX107 (pGUS-PWT77).
C. Mutational analysis of the promoter region. b-Glucuronidase activities of R. etli CFNX107 transconjugants containing mutations in the promoter
region of a repA::gusA fusion: bar 1, CFNX107 (pBBMCS53); bar 2, CFNX107 (pGUS-PWT95); bar 3, CFNX107 (pGUS-PMB10); bar 4, CFNX107
(pGUS-PMB35); bar 5, CFNX107 (pGUS-PCS35 Ec); bar 6, CFNX107 (pGUS-PCS3510 Ec); bar 7, CFNX107 (pGUS-PEM).
D. Functionality of the repA promoter region and its mutant derivatives in E. coli. b-Glucuronidase activities of R. etli CFNX107 containing the
promoterless gusA vector (bar 1) and with plasmid (pGUS-PWT95) (bar2) as controls, compared with b-glucuronidase activities of E. coli strains: bar
3, BW21038 (pGUS-PWT95); bar 4, BW21038 (pGUS-PCS35 Ec); and bar 5, CFNX107 (pGUS-PCS3510 Ec). Specific activities are reported as
nmol min21 mg21 protein. Each value is the average of at least three independent experiments

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204

198 M. A. Ramı́rez-Romero

constructs were transferred into an R. etli strain CFNX107

(pGUS-PWT160) to provide in trans several combinations
of the proteins encoded in the pSym replicator region. The
constructs introduced were: (i) pKRE-1, containing the
complete repABC operon; (ii) pKRE-prepAB, harbouring
functional repAB genes; (iii) pKRE-prepDA-BC, carrying a
repABC operon with an in frame deletion in repA; (iv)
pKRE-prepA-DB-C, containing a repABC operon with an in
frame deletion in repB; (v) pKRE-DA1, carrying a repABC
operon in which the repAB genes were deleted; and (vi)
pKRE-prepA, with a functional repA gene. The b-
glucuronidase activity of each transconjugant strain was
determined. As shown in Fig. 3, constructs containing
a functional repA gene repressed the expression of
repA::gusA (pGUS-PWT160), whereas those without a
functional repA express b-glucuronidase at the same level
as the repA::gusA (pGUS-PWT160) alone. These results
show that RepA is a negative transcriptional regulator of Fig. 2. Determination of the 50 end of the repABC transcript by primer
extension. The RNA for the primer extension reaction was obtained
the repABC operon. RepB and RepC do not play a from strain CFNX107 (pGUS-PWT160). Lanes C, T, A and G are
significant role in this function. sequencing reactions of plasmid pGUS-PWT160 performed with the
same oligonucleotide used in the primer extension reaction. The
primer extension product is marked with an asterisk. The relevant
Localization of the RepA operator site DNA sequence and the nucleotide 11 are shown to the right.

To determine the sites of interaction between RepA and

the repABC promoter region, an in vivo DMS footprinting
analysis was made using two R. etli strains: a pSym-
cured derivative carrying the repA::gusA construct
(CFNX107/pGUS-PWT160) and the same strain plus
pKRE-prepA. The results of this analysis are shown in
Fig. 4 and indicate that RepA protects three G nucleotides
located at positions 14, 122 and 127 relative to the
transcriptional start. Two of these nucleotides lie within
inverted repeats of 14 bp located between the putative
promoter region of the repABC operon and the initial
codon of repA.
To support the view that this region contains the RepA
operator site, the DNA region located between position 12
to 128 of the repA::gusA of plasmid pGUS-PWT95 was
deleted and introduced into R. etli CFNX107, where this
deletion derivative (pGUS-PMIR) expressed b-glucuroni-
dase at the same level as the parental plasmid. However,
when it was mobilized into strain CFNX107/pKRE-prepA,
no reduction in the expression of b-glucuronidase was
Fig. 3. Identification of the transcriptional negative regulator. The b-
observed, indicating that pGUS-PMIR is insensitive to the glucuronidase activity of: R. etli with the symbiotic plasmid (CFNX
presence of RepA (see Fig. 5). This genetic analysis is 101) and carrying the promoterless gusA vector (bar 1) or carrying
consistent with the proposition that the RepA operator site pGUS-PWT95 (bar 2). Bars 3 and 4 represent the b-glucuronidase
activities of an R. etli strain cured of its symbiotic plasmid (CFNX107)
includes position 12 to 128. but containing the promoterless gusA vector (bar 3) or plasmid
(pGUS-PWT95) (bar 4). The next bars represent the b-glucuronidase
activities of derivatives of R. etli strain CFNX107 (pGUS-PWT95)
Transcription is not the only barrier limiting the replication carrying: bar 5, pKRE-1 (A1, B1, C1); bar 6, pKRE-prepAB (A1, B1,
of the R. etli symbiotic plasmid in E. coli C – ); bar 7, pKRE-prepDA-BC (A – , B1, C1); bar 8, pKRE-prepA-DB-C
(A1, B – , C1); bar 9, pKRE-DA1 (A – , B – , C1); bar 10, pKRE-prepA
To test whether the 5.6 kb HindIII fragment containing (A1, B – , C – ). Specific activities are reported as nmol min21 mg21
protein. Each value is the average of at least three independent
the basic replicon of the R. etli pSym was able to replicate experiments. Bars with an asterisk are the b-glucuronidase activities
in E. coli, this fragment was cloned in pMAK705, a of strains expressing RepA.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204


temperature-sensitive pSC101 replicon derivative, and
transformed into E. coli. Transformants were unable to
grow at the non-permissive temperature in the presence of
the selective marker. This result suggests that the pSym
basic replicon is unable to replicate in E. coli.
As described above, the promoter of the repABC operon
is not functional in E. coli and, thus, presents a barrier
for the replication of the R. etli symbiotic plasmid in E. coli.
To overcome this problem, the promoter of the repABC
operon was exchanged for the lac promoter of plasmid
pBluescript-II SK1, and an E. coli Shine – Dalgarno
sequence (AAGGAA) was also placed 6 bp upstream of
the repA initial codon. HindIII restriction sites flanked the
new chimeric replicon.
To test its functionality in R. etli, the chimeric replicon
was cloned in pSUP202, a vector that is unable to replicate
in R. etli. This construct was introduced by conjugation into
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204
RepA is the negative regulator of the repABC operon 199
Fig. 4. Genomic DMS footprinting of the R. etli
repABC promoter region. The repA upstream
region of plasmid pGUS-PWT160, carrying a
repA::gusA fusion, was analysed by DMS
footprinting in two genetic backgrounds, one
expressing RepA (CFNX107/pKRE-prepA) and
one not expressing repA (CFNX107). Primer
extension products obtained by linear
amplification of the genomic DNA with Taq
polymerase were separated by gel
electrophoresis and detected with a Molecular
Dynamics PhosphorImager device. Positions of
the protected G nucleotide residues are
indicated by arrows. Regions containing RepA-
protected G residues (I, II, III) were scanned
and compared with the equivalent region in the
strain not expressing RepA. The normalized
scans of each region are shown in (B).
R. etli CFNX107. Analysis of the plasmid profiles of the
transconjugants demonstrated that the chimeric replicon is
maintained as an independent molecule. To examine
whether the chimeric replicon is able to replicate in E. coli,
it was cloned in pMAK705 to generate pMAK-PlacrepABC
and transformed into E. coli. At the permissive tempera-
ture (308C), the construct replicated in E. coli but, at non-
permissive temperature (448C), the plasmid is rapidly
lost, indicating that the R. etli chimeric replicon was non-
functional in this organism. This result shows that
transcription is not the only barrier limiting the replication
of the R. etli symbiotic plasmid in E. coli.
Discussion
A unique feature of the members of the repABC family is
that their replication and partition proteins are encoded in a
200 M. A. Ramı́rez-Romero
Fig. 5. Localization of the RepA operator region. b-Glucuronidase
activities of R. etli CFNX107 transconjugants containing repA::gusA
fusions, with and without the proposed RepA operator region, and its
behaviour in the presence or absence of RepA (see Fig. 1A). Bar 1,
CFNX107 carrying the promoterless gusA vector; bar 2, CFNX107
(pGUS-PWT95); bar 3, CFNX107 (pGUS-PWT95)(pKRE-prepA); bar
4, CFNX107 (pGUS-PMIR); bar 5, CFNX107 (pGUS-PMIR)(pKRE-
prepA). Specific activities are reported as nmol min21 mg21 protein.
Each value is the average of at least three independent experiments.
Bars with an asterisk are the b-glucuronidase activities of strains
expressing RepA.
single operon. In other large, low-copy-number plasmids,
such as the F, P1 and P7 plasmids, the replication and
partition functions are encoded in different loci and, as a
consequence, each of these sets of genes is regulated
independently. In these plasmids, the initiator protein
negatively regulates its own transcription by binding to
repetitive sequences (iterons) that overlap the promoter
(Chattoraj, 2000). Autoregulation of the partition operons
is a common theme in low-copy-number plasmids. In
particular, in the F, P1 and P7 plasmids, the partition
proteins (A and B) act together, autoregulating their own
transcription. This observation is of special interest
because it has been shown that the repA and B genes of
the pSym of R. etli CE3 are structurally and functionally
homologous to the A and B polypeptides of the par/sop
partition systems of plasmids F, P1 and P7. To elucidate
the mechanism of transcriptional regulation of the repABC
operon of the R. etli symbiotic plasmid, the transcription
start site was determined, and the promoter region of this
operon was delimited.
The transcription start site (11) of the repABC operon
was located 45 bp upstream of the initiation codon of repA,
as determined by primer extension. Two lines of evidence
indicate that the repABC operon promoter was identified
correctly: (i) a sequence similar to the E. coli s70 promoter
consensus was located 7 bp upstream of the transcrip-
tional start site; and (ii) mutations altering the proposed
210 or 235 hexameric elements of the repABC operon
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204
promoter destroy its function, but not those located in the
spacer region located between these two elements.
When a repA::gusA carrying the repABC wild-type
promoter (pGUS-PWT160) was introduced into E. coli, no
b-glucuronidase activity could be detected, indicating
that the promoter is not functional in E. coli. This was a
predictable result, because mutations that change the
E. coli promoter consensus in the 235 element from
[TTGACA] to [TTGCCA] or from [TTGACA] to [TTGATA]
severely diminish promoter function (McClure et al., 1983).
The 235 element of the repABC promoter contains these
two changes, making it a non-functional 235 element in
E. coli.
A compilation of E. coli mRNA promoter sequences
indicates that nucleotide ‘A’ in the second position (211)
on the 210 hexameric element is conserved in 76% of the
promoters (Lisser and Margalit, 1993). A change from
[TATAAT] to [TCTAAT] results in a severe reduction in
promoter activity in E. coli, and this is precisely the 210
element found in the promoter of the R. etli repABC operon
(Moyle et al., 1991).
The introduction of nucleotide changes in the 210 and
235 hexameric elements of the R. etli repABC promoter to
match the E. coli consensus resulted in a promoter that
was functional in E. coli and in R. etli.
One novel finding of this work is that the repABC operon
is regulated negatively by RepA, and that RepB and RepC
do not participate in the regulation of their transcription.
This is the first case described in which a partition protein
regulates the transcription of an initiator protein.
An in vivo DMS footprinting indicates that RepA protects
a region between positions 12 to 128 relative to the
transcriptional start site. Moreover, an R. etli derivative
carrying a repA::gusA fusion that lacked the DNA between
positions 12 to 128 (pGUS-PMIR) was capable of
expressing b-glucuronidase at the same level as the
wild-type repA::gusA fusion, but it was not subject to
repression in the presence of a source of RepA in trans.
These results are in agreement with the proposition that
the RepA operator lies between nucleotide residues 12
and 128, a position that is compatible with its role as a
repressor.
The DNA sequence between positions 12 and 118 is
almost identical to that found in the upstream region of
the repABC operon of pRiA4b, but is absent in the other
sequenced RepABC plasmids, suggesting that the
interaction of RepA with its operator is plasmid specific.
Precise determination of the RepA operator region will
require DNase I footprinting experiments in vitro with the
purified RepA.
To take advantage of the genetic and genomic tools
available in E. coli, the replication capacity of the R. etli
basic replicon was tested in this species. The wild-type
basic replicon or a derivative, in which a lacZ promoter was
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204 Table 2. Strains and plasmids used in this work.

Strain or plasmid Relevant propertiesa Reference

Strain
Escherichia coli
HB101 Host strain for plasmids Boyer and Roulland-Dussoix (1969)
DH5a Host strain for plasmids Hanahan (1983)
BW21038 gusA derivative Metcalf and Wanner (1993)
Rhizobium etli
CE3 Smr, nodulates P. vulgaris Noel et al. (1984)
CFNX101 recA::V, Spr/Smr, derivative of CE3 Martı́nez-Salazar et al. (1991)
CFNX107 recA::V, Spr//Smr, p42a – , p42d – derivative of CE3 Martı́nez-Salazar et al. (1991)
Plasmid
pSUP202 Apr Cmr Tcr, ColE1 origin unable to replicate in Rhizobium spp. Simon et al. (1983)
pRK7813 Tcr, RK2-based plasmid vector Jones and Gutterson (1987)
pRK2013 Helper plasmid, Kmr Ditta et al. (1980)
pWM5 b-Glucuronidase gene donor Metcalf and Wanner (1993)
pBBR1MCS-5 Gmr cloning vector replicable in Rhizobium Kovach et al. (1995)
pBBMCS53 pBBR1MCS-5 derivative carrying a promoterless b-glucuronidase gene Corvera et al. (1999)
pMAK705 Cmr, temperature-sensitive pSC101 replicator Hamilton et al. (1989)
pKRE-1 pRK7813 derivative with the 5.6 kb HindIII fragment encoding Ramı́rez-Romero et al. (2000)
the basic replicon of the pSym of R. etli CE3
pKRE-prepDA-B pRK7813 derivative containing repB and an in frame deletion in the repA gene Ramı́rez-Romero et al. (2000)
pKRE-prepA-DB pRK7813 derivative containing repA and an in frame deletion in the repB gene Ramı́rez-Romero et al. (2000)
pKRE-prepA pRK7813 derivative containing repA and its upstream region Ramı́rez-Romero et al. (2000)
pKRE-prepAB pRK7813 derivative containing the complete repAB genes Ramı́rez-Romero et al. (2000)

RepA is the negative regulator of the repABC operon

with its upstream region
pGUS-PWT160 A pBBMCS53 derivative containing 160 bp upstream of the initial codon of repA and This work
264 bp of the coding region of repA transcriptionally fused to the b-glucuronidase gene
pGUS-PWT95 A pBBMCS53 derivative containing 95 bp upstream of the initial codon of repA and This work
264 bp of the coding region of repA transcriptionally fused to the b-glucuronidase gene
pGUS-PWT82 A pBBMCS53 derivative containing 82 bp upstream of the initial codon of repA and This work
264 bp of the coding region of repA transcriptionally fused to the b-glucuronidase gene
pGUS-PWT77 A pBBMCS53 derivative containing 77 bp upstream of the initial codon of repA and This work
264 bp of the coding region of repA transcriptionally fused to the b-glucuronidase gene
pGUS-PMIR A pGUS-PWT95 derivative carrying a deletion between This work
nucleotides 18 and 44 upstream of the initial codon of repA
pGUS-PMB10 A pGUS-PWT95 derivative in which the promoter hexameric 210 This work
element (TCTAAT) was changed to (TCGCCT)
pGUS-PMB35 A pGUS-PWT95 derivative in which the promoter hexameric 235 This work
element (TTGCTC) was changed to (TGTTTC)
pGUS-PC35Ec A pGUS-PWT95 derivative in which the promoter hexameric 235 This work
element (TTGCTC) was changed to match the E. coli consensus (TTGACA)
pGUS-PC3510Ec A pGUS-PWT95 derivative in which promoter hexameric 235 This work
and 210 elements were changed to match the E. coli consensus
pSUP-PlacrepABC A pSUP202 carrying the repABC genes without their own promoter sequences This work
but under the lac promoter of plasmid pBluescript-II SK1. Also, an E. coli Shine – Dalgarno
sequence (AAGGAA) was placed 6 p upstream the repA initial codon
pMAK-PlacrepABC A pMAK705 derivative carrying the same insert as plasmid pSUP-PlacrepABC This work
pGUS-PEM A pGUS-PWT95 derivative in which the region located between the 235 and 210 This work
elements was changed from (AAAATGCAGAATCGGC) to (TGCATGCAGAATGAGA)

201
202 M. A. Ramı́rez-Romero
substituted the repA upstream sequence, was unable to
replicate in E. coli, indicating that transcription is not the
only barrier to the replication of the RepABC plasmids in
E. coli. It remains to be tested whether the repABC
proteins are translated in E. coli (the repABC operon does
not contain classical E. coli Shine –Dalgarno sequences)
and whether repABC proteins can recruit the replication
machinery of E. coli.
Experimental procedures
Bacterial strains and growth conditions
Bacterial strains and plasmids used in this work are listed in
Table 2. E. coli strains were grown at 378C in Luria –Bertani
medium. Rhizobium strains were grown at 308C in PY medium
(Noel et al., 1984). Antibiotics were added at the following
concentrations (in mg ml21): gentamicin 30; tetracycline 10;
chloramphenicol 25; carbenicillin 100; nalidixic acid 20.
Bacterial matings
pSUP202 or pRK7813 derivatives were introduced into
Rhizobium using pRK2013 as helper plasmid. Strains were
grown in the proper liquid medium to stationary phase, mixed
in a proportion of donor– helper–receptor of 1:1:2 on PY
plates and incubated at 308C overnight. Cells were
resuspended in fresh PY medium, and serial dilutions were
plated on the appropriate selective medium.
DNA isolation, manipulation and hybridization
Genomic DNA was isolated using components and instruc-
tions from a DNA/RNA isolation kit (Amersham). Plasmid DNA
was isolated as described by Sambrook et al. (1989). DNA
was restricted and ligated under the conditions specified by
the enzyme manufacturer (Amersham). Taq DNA polymerase
or Elongase (Gibco BRL) was used for polymerase chain
reaction (PCR). The PCR products were cloned using a
pMOSblue T vector kit or a pMOSblue blunt-ended kit
(Amersham).
RNA isolation
RNA was isolated using components and instructions from the
Perfect RNA kit (5 Prime-3 Prime).
Plasmid profiles
Profiles of high-molecular-weight plasmids were obtained by
the in-gel lysis procedure described by Wheatcroft et al. (1990).
Primer extension
To determine the transcriptional start site of the repABC
operon, oligonucleotide (repA30) 50 -ACGACCGGCGCT
GTGTTT-30 , which is complementary to the DNA region
located between position 15 and 32 from the initiation codon,
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 195–204
was used. Primer extension was carried out using 12 mg of
RNA isolated from strain CFNX107 (pGUS-PWT160).
Oligonucleotides were annealed with the RNA in 0.12 M
NaCl20.25 M Tris-HCl (pH 7.0) by heating for 3 min at 958C
and then slowly cooling to 428C. Primer extensions were
carried out at 428C for 1 h with avian myeloblastosis virus
reverse transcriptase in transcription buffer [50 mM Tris-HCl,
50 mM KCl, 10 mM MgCl2, 5 mM dithiothreitol (DTT), pH 7.5],
1 mM deoxynucleoside triphosphates, 100 mM DTT and
108 U of RNasin (Amersham). The extension products were
precipitated with 2.5 M ammonium acetate and ethanol and
analysed by electrophoresis on 6% polyacrylamide gels. A
ladder was obtained by sequencing plasmid pGUS-PWT160
with the same oligonucleotide and run during the electrophor-
esis, side by side with the primer extension sample. The gels
were scanned in a Molecular Dynamics PhosphorImager
(Morett and Buck, 1988).
Genomic DMS footprinting
DMS was added to a final concentration of 0.1% to 5 ml
cultures of the desired strains grown at an OD540 of 0.4. After
1 min, cells were collected by centrifugation and washed twice
with saline phosphate solution. The methylated DNA was
purified and cleaved by incubation with piperidine. The
methylation pattern was obtained by primer extension using
8 mg of DNA, 0.5 pmol of repA30 oligonucleotide labelled and
extended by linear amplification using Taq DNA polymerase
(20 cycles). The products were precipitated with 2.5 M
ammonium acetate and ethanol and analysed by electro-
phoresis on 6% polyacrylamide gels. The gels were scanned
in a Molecular Dynamics PhosphorImager device (Morett and
Buck, 1988).
Construction of the repA::gusA fusions
To determine the minimal region required for transcription of
the repABC operon, PCRs containing repA upstream regions
of several lengths and the first 160 nucleotides of the coding
region of repA were transcriptionally fused to the promoterless
gusA gene of plasmid pBBMCS53. To do this, the PCR
oligonucleotides of the repA upstream regions were designed
to introduce an Xba I restriction site in its 50 end. The
oligonucleotide complementary to the coding sequence of
repA includes a Sal I restriction site in its 50 end. The different
PCR products were cloned in plasmid pBBMCS53 using the
introduced Xba I and Sal I restriction sites, which are also
present in the vector.
The different repA::gusA fusions were introduced to R. etli
CFNX107 by triparental mating, and their b-glucuronidase
activities were determined as described above.
Mutant versions of the promoter region found in plasmid
pGUS-PWT160 were constructed by PCR using oligonucleo-
tides carrying altered versions of the repA upstream region
and the same oligonucleotides complementary to the coding
sequence of repA described above.
b-Glucuronidase activity measurements
Overnight cultures of the strains carrying the desired

constructs were grown in the presence of the appropriate
selection. Cultures were diluted in fresh PY medium to an
OD540 of 0.01 and grown to a final OD540 of 0.4. Culture (1 ml)
was centrifuged and resuspended in a salt wash solution
supplemented with chloramphenicol (100 mg ml21). Quanti-
tative b-glucuronidase assays were performed with p-nitro-
phenyl glucuronide as substrate as described previously
(Wilson et al., 1992). Data were normalized to total-cell
protein concentration by the Lowry method (Sambrook et al.,
1989). The results presented in the figures are the mean of
three independent experiments.
Construction of the lacZ –repABC chimeric replicon
To exchange the upstream region of the repABC operon for
the lacZ promoter, a PCR was used to amplify the repABC
operon from the initial codon of repA to 500 bp downstream of
repC. One of the oligonucleotides used in this PCR adds an E.
coli Shine– Dalgarno and a Bam HI restriction site at the repA
end of the PCR product. The other one adds a HindIII
restriction site 500 bp downstream of repC. This PCR product
was restricted with Bam HI and HindIII and cloned in
pBluescript-II SK1 using the same restriction sites. The
procedure orients the repABC PCR product in the same
direction as the transcription of the lacZ gene of the vector.
Later, the insert was amplified using the same oligonucleotide
located 500 bp downstream of repC and an oligonucleotide
located 154 bp upstream of the initial codon of the lacZ gene
of the pBluescript-II SK1 vector, which also adds a HindIII
restriction site at the 50 end. This PCR amplifies the repABC
genes with a lacZ promoter at its 50 end and adds HindIII
restriction sites at both sides of the product. The second PCR
product was restricted with HindIII and cloned in pSUP202
and pMAK705 to generate constructs pSUP-PlacRepABC
and pMAK-PlacRepABC respectively.
Replication test in E. coli
To test the replication capacity of the pSym repABC
derivatives in E. coli, they were cloned in the HindIII restriction
site of pMAK705. The constructs were transformed into E. coli
DH5a, plated in selective media and incubated at 308C.
Transformants were grown overnight at 308C in 5 ml of LB with
the appropriate antibiotic, diluted and plated on prewarmed
selective plates and incubated at 308C or 408C (Hamilton et al.,
1989).
Acknowledgements
We wish to thank Michael Dunn, Lorenzo Segovia and David
Romero for their critical comments, and Rosa Angélica Rivas
for her skilful technical support. We also thank Paul Gaytán
and Eugenio López for the synthesis of oligonucleotides. This
work was supported by CONACyT grant 27850N and DGAP-
PAPIIT grant IN214898.
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