Vol. 20, No. 7, 2007 / 867
MPMI Vol. 20, No. 7, 2007, pp. 867–881. doi:10.1094/MPMI-20-7-0867. © 2007 The American Phytopathological Society
The Rhizobium leguminosarum bv. trifolii RosR:
Transcriptional Regulator Involved
in Exopolysaccharide Production
Monika Janczarek and Anna Skorupska
Department of General Microbiology, University of M. Curie-Skłodowska, Akademicka 19, 20-033 Lublin, Poland
Submitted 19 October 2006. Accepted 4 March 2007.
The acidic exopolysaccharide is required for the establish-
ment of symbiosis between the nitrogen-fixing bacterium
Rhizobium leguminosarum bv. trifolii and clover. Here, we
describe RosR protein from R. leguminosarum bv. trifolii
24.2, a homolog of transcriptional regulators belonging to
the family of Ros/MucR proteins. R. leguminosarum bv.
trifolii RosR possesses a characteristic Cys2His2 type zinc-
finger motif in its C-terminal domain. Recombinant
(His)6RosR binds to an RosR-box sequence located up-
stream of rosR. Deletion analysis of the rosR upstream
region resulted in identification of two –35 to –10 promoter
sequences, two conserved inverted palindromic pentamers
that resemble the cAMP-CRP binding site of Escherichia
coli, inverted repeats identified as a RosR binding site, and
other regulatory sequence motifs. When assayed in E. coli,
a transcriptional fusion of the cAMP-CRP binding site
containing the rosR upstream region and lacZ gene was
moderately responsive to glucose. The sensitivity of the
rosR promoter to glucose was not observed in E. coli
Δ ΔcyaA. A rosR frame-shift mutant of R. leguminosarum bv.
trifolii formed dry, wrinkled colonies and induced nodules
on clover, but did not fix nitrogen. In the rosR mutant,
transcription of pssA-lacZ fusion was decreased, indicating
positive regulation of the pssA gene by RosR. Multiple cop-
ies of rosR in R. leguminosarum bv. trifolii 24.2 increased
Additional keywords: catabolic repression, zinc finger protein.
Rhizobium leguminosarum produces large amounts of acidic
exopolysaccharide (EPS) that has been shown to be indispen-
sable for nodule invasion and, therefore, for successful nitro-
gen-fixing symbioses with legumes that form an indeterminate
type of nodules with persistent meristem, such as Medicago,
Pisum, Trifolium, and Vicia spp. (Becker and Pühler 1998;
Perret et al. 2000). EPS-deficient mutants of Rhizobium legu-
minosarum fail to nodulate their host plants (Borthakur et al.
1988) or induce formation of ineffective nodules (Rolfe et al.
1996, Skorupska et al. 1995; van Workum et al. 1997). The
EPS of R. leguminosarum is a polymer of octasaccharide sub-
units, each composed of one galactose, five glucose, and two
glucuronic acid residues, additionally decorated with acetyl,
pyruvyl, and hydroxy-butanoyl modifications (Hollingsworth
et al. 1988). EPS production requires the expression of several
exo or pss genes. Most of them are dispersed throughout the
genome, except for three major EPS clusters of chromosomal
genes of R. leguminosarum bv. viciae (Young et al. 2006) and
R. etli (González et al. 2006). The largest pss gene cluster
includes the pss genes encoding specific glycosyl transferases,
epimerases, and deacetylases involved in the biosynthesis of
the EPS repeating unit, genes encoding proteins engaged in
polymerization and transport of EPS, and other genes encoding
enzymes that modify EPS (González et al. 2006; Skorupska et
al. 2006). Most of these genes and their specific roles in EPS
production have not been studied to date.
Unlike with Sinorhizobium meliloti, knowledge of regulation
of pss gene expression in R. leguminosarum is only fragmen-
tary. Previously, the psi and psr regulatory genes have been
described in R. leguminosarum bv. phaseoli (now R. etli)
(Lachford et al. 1991; Mimmack et al. 1994a,b). Even though
a psiA mutant was not altered in its ability to produce EPS, it
elicited empty nodules without infection threads and bacteria
on Phaseolus spp. (Borthakur and Johnston 1987; Lachford et
al. 1991). The increased copy number of psiA prevented the
production of EPS in R. leguminosarum bvs. phaseoli and
viciae and abolished nodulation ability of their respective
hosts. The inhibitory effect of multiple copies of psiA on nodu-
lation and EPS production could be overcome in the presence
of additional copies of psr or pssA encoding the first glucosyl-
IP-transferase (Lachford et al. 1991; Mimmack et al. 1994a,b).
An exoR regulatory gene with extensive similarity to exoR of
S. meliloti has been identified on the chromosome of R. legu-
minosarum bv. viciae (Reeve et al. 1997). An exoR mutant pro-
duced three times as much EPS as the wild-type strain, which
suggested the role of this gene in negative regulation of EPS
An rosR gene encoding a transcriptional regulatory protein
has been identified in R. etli CE3 (Araujo et al. 1994, Bittinger
et al. 1997). An rosR deletion mutant formed mucoid, domed
colonies, efficiently nodulated bean plants, and produced EPS
that was identical to that produced by the wild-type strain.
However, the mutant was significantly impaired with respect to
nodulation competitiveness and nodule occupancy compared
with the parental strain (Bittinger et al. 1997). R. etli RosR
regulates more than 50 genes affecting the expression of many
functionally diverse genes, as revealed by genome-wide genetic
screening (Bittinger and Handelsman 2000).
RosR of R. etli shares significant identity with the Ros protein
of Agrobacterium tumefaciens (Chou et al. 1998). Ros protein
recognizes and binds the so-called ros-box, a 40-bp A:T-rich
sequence located within the operators of virC and virD and
Corresponding author: A. Skorupska; Telephone: +48 81 537 59 72, Fax:
+48 81 537 59 59; E-mail: firstname.lastname@example.org
Nucleotide and amino acid sequences are available in GenBank database
under accession number AY683453 and AY683454.
868 / Molecular Plant-Microbe Interactions
upstream of the ipt gene. Ros negatively regulates virC and
virD while positively affecting EPS production. Mutations in
the ros gene result in derepression of virC and virD and the
loss of succinoglycan production (Chou et al. 1998).
RosR of R. etli also displays high amino acid sequence simi-
larity with MucR, which acts as a transcriptional repressor of
galactoglucan (EPS II) production and an activator of suc-
cinoglycan (EPS I) production in S. meliloti 2011 (Becker et
al. 1997; Keller et al. 1995). The mucR mutant produced EPS
II, whereas only very small amounts of low molecular weight
EPS I oligosaccharides were secreted (Keller et al. 1995; Zhan
et al. 1989). MucR repressed exp genes encoding EPS II bio-
synthesis enzymes but had a weak or no effect on transcription
of the exo genes that encode EPS I synthesis proteins (Bertram-
Drogatz et al. 1998; Glazebrook and Walker 1989; Keller et al.
1995; Quester and Becker 2004). However, in S. meliloti EFB1,
MucR is essential for galactoglucan production and mucR mu-
tants are completely nonmucoid (Martin et al. 2000).
The C-terminal part of MucR/Ros proteins contains an amino
acid sequence that reveals similarity to the Cys2His2-type zinc-
finger motif, which was found primarily in DNA-binding pro-
teins of eukaryotic origin (Berg and Shi 1996; Cooley et al.
1991). Four conserved residues are ligands for a central zinc
ion that stabilizes small globular domain (i.e., the finger do-
main) (Pabo and Sauer 1992). Eukaryotic transcription factors
using this motif typically contain tandem arrays of such C2H2
zinc fingers that bind to neighboring sites on the DNA. Each
finger has a conserved αββ structure and the amino acids on
the surface of the α-helix contact the bases in the major
groove. MucR/Ros proteins contain only one zinc-finger motif.
In Ros of A. tumefaciens, the peptide loop spacing of the zinc
finger is 9 amino acids (aa) as opposed to the invariant 12 aa in
the classical C2H2 motif (Chou et al. 1998).
In this study, we present the results of rosR gene identifica-
tion in R. leguminosarum bv. trifolii strains 24.2 and TA1. The
rosR gene undergoes a complex transcriptional control, as evi-
denced by mapping of several regulatory sites in the upstream
region of rosR and deletion analysis of this region. The rosR
gene is subject to negative autoregulation by its own protein
product. Using an electrophoretic mobility shift assay (EMSA),
we assayed binding of the purified (His)6RosR protein to an
RosR-box sequence identified in the rosR upstream region.
The rosR mutant formed dry, wrinkled colonies and elicited
nodules on clover plants, but did not fix nitrogen.
Cloning of rosR gene and sequence analysis.
Using the genomic sequence of R. etli (Bittinger et al. 1997)
as a guide in primer design, the rosR homolog was isolated
from R. leguminosarum bv. trifolii 24.2 and TA1 strains by po-
lymerase chain reaction (PCR) amplification. After amplifica-
tion by PCR, rosR genes of 24.2 and TA1 strains were cloned
into pUC19 to form the pB31 and pB35 plasmids, respectively,
and the 1,174-bp insert of pB31 and 856-bp insert of pB35
were sequenced. In both cases, sequence analysis indicated the
presence of a 432-bp-long open reading frame (ORF) with a
putative ribosome binding site (AGGAGA) located 6 bp up-
stream of the potential ATG translation start codon (Fig. 1).
Downstream of the TGA stop codon, a palindrome sequence of
rho-independent transcription termination sites was found. The
sequences of both rosR ORFs were identical; therefore, in fur-
ther study, only the rosR gene of Rt24.2 strain was analyzed.
The predicted protein product of the rosR coding region was
composed of 143 aa (15.7 kDa) and was highly similar to R.
etli RosR (98% identity at the protein level), S. meliloti MucR
(80% identity), A. tumefaciens Ros (80% identity), A. radio-
bacter RosAR (80% identity), and Rhizobium sp. strain
NGR234 MucR (75% identity). Homologs of the rosR gene
also have been found in other rhizobia (e.g., Mesorhizobium
loti MAFF303099 [accession nos. BAB54066 and BAB52589]
BAC48333]). Southern hybridization with the rosR gene as a
probe showed one hybridization site, indicating a single rosR
locus in Rt24.2 and RtTA1 strains (data not shown).
As with other proteins of the Ros/MucR subfamily, a puta-
tive zinc-finger domain was found at the carboxy-terminal half
of the RosR protein of Rt24.2. It was composed of 19 aa resi-
dues, including two conserved cysteines (C) and two conserved
histidines (H) in a C-N2-C-N9-H-N3-H-type motif (Fig. 1). In
this type of regulatory protein, one atom of Zn is tetrahedrally
coordinated by the conserved C and H residues composing the
finger, which interacts with short runs of guanine residues in
DNA (Chou et al. 1998).
Functional analysis of rosR upstream region.
Computer analysis of the rosR upstream region revealed the
presence of several regulatory sequences, including the RosR-
box (position –133 to –112 bp relative to the start codon) (Fig.
1). The RosR-box is a 22-bp-long sequence containing two
nine-nucleotide inverted repeats separated by four G and is the
consensus sequence identified as the binding site for MucR/
Ros transcriptional regulators in rhizobia (Bertram-Drogatz et
al. 1997; Keller et al. 1995). This sequence is identical to the
Ros-box of R. leguminosarum bv. viciae (Young et al. 2006),
almost identical to R. etli (Bittinger et al. 1997), and similar to
the Ros-box of A. radiobacter (Brightwell et al. 1995) and A.
tumefaciens (Cooley et al. 1991). The sequence of the MucR-
box in S. meliloti (Keller et al. 1995) is most divergent (Fig. 2).
Upstream of the rosR coding region, a putative promoter
with a score of 0.95 (position –355 to –278 bp) was identified.
In the in silico predicted sequence, two putative promoters (P1
and P2), highly similar to the eubacterial sigma factor (σ70)
binding site (Wösten 1998), were found: one –35 and –10 con-
sensus sequence with an optimal spacing of 17 bp (5′-TTGGC
GN17TATTTG), and another –35 and –10 putative promoter
with longer spacing between two hexamers (5′ATGCAAN20TA
CAAT) (Fig. 1). Apart from the promoters, at position –169 to
–125 relative to the start codon, three repeats of a conserved T-
(N11)-A element were found. The element comprises a LysR
motif present in many promoters regulated by LysR-type tran-
scriptional regulators (Schell 1993), among them the NodD
protein that positively controls the synthesis of the nodulation
factor in rhizobial symbioses (Long 2001) and SyrM protein
that belongs to the family of transcriptional activators of EPS I
production in S. meliloti (Dusha et al. 1999). The third T-(N11)-
A repeat partially overlaps the RosR-box.
To functionally analyze the putative rosR promoter, a set of
plasmids with 5′-end (pEP0-pEP7) and 3′-end deletions
(pEP8-pEP12) of the promoter region fused to a promoterless
lacZ gene were constructed (Fig. 3). E. coli DH5α cells were
transformed with these plasmids, which subsequently were
transferred conjugally to the wild-type Rt24.2 strain. β-galac-
tosidase activities in DH5α and Rt24.2 strains carrying plas-
mids with different lacZ fusions were assayed (Fig. 3). The
pEP0 plasmid containing the longest promoter fragment re-
vealed a significantly lower activity in DH5α and slightly
lower activity in Rt24.2 when compared with the next tran-
scriptional fusion (pEP1). Starting from –357 bp (pEP2), the
progressive 5′-end deletions reduced the rosR expression level
in both genetic backgrounds (Fig. 3). In E. coli, a nearly three-
fold reduction of LacZ activity was observed for the –281 fu-
sion (plasmid pEP3) relatively to the –403 fusion (pEP1), and
then a gradual decrease of activity up to the –219 fusion
Vol. 20, No. 7, 2007 / 869
(pEP4). In Rt24.2, the reduction of LacZ activity for the –281
fusion (pEP3) was more drastic (approximately 6.5 times) and,
for the –235 fusion (pEP3C), the LacZ activity was practically
at the background level. Overall, deletion of the two putative
promoters containing the –35 to –10 consensus elements (Fig. 1)
resulted in complete reduction in β-galactosidase activity in
When the rosR upstream sequence (Fig. 1) was further ana-
lyzed, two sequences highly similar to cAMP-CRP binding
sites in E. coli (Ebright et al. 1989) were identified. Specific
Fig. 1. Nucleotide sequence of 1,174-bp fragment containing rosR gene of Rhizobium leguminosarum bv. trifolii 24.2 with the upstream region. The deduced
amino acid sequence of rosR is given in the single-letter code. The putative C2H2 motif in RosR protein, RosR-box, and cAMP-CRP binding sites are shaded
in light gray. P1 and P2 –35 and –10 promoter sequences are marked by dark-gray shaded boxes. Transcription start sites are marked by an angled arrows.
Putative promoters and palindromic terminator sequences are underlined. The ribosome binding site (rbs), translation start codon, and short, direct repeats
(DR) are boxed. Long inverted repeats (IR1 and IR2) are overlined with arrows. LysR motifs are underlined and marked with stars. Overlined short arrows
indicate location of primers used for polymerase chain reaction amplification of DNA fragments of the upstream rosR region. Shaded box in +177 position of
rosR indicates deletion of C nucleotide resulting in a frame-shift mutation.
870 / Molecular Plant-Microbe Interactions
binding sites for cAMP-CRP usually are located upstream of
the promoters and are essential for transcriptional activation or
repression. In the case of rosR, the first imperfect inverted pal-
indromic sequence, GC-TGTGA-N7-TTGCT-GG, was located
upstream of the P1 promoter. The second cAMP-CRP binding
site (i.e., AT-TGTGA-N7-TCGTA-CT) was found downstream
of the P1 promoter and partially overlapped the –10 sequence
of P2 (Fig. 1). It was identical (first pentamer) or highly simi-
lar (second pentamer) to the consensus sequence AAN-
TGTGA-N6-TCACA-T, which is responsible for binding the
CRP dimer in complex with cAMP in E. coli, resulting in the
regulation of transcription (Kolb et al. 1993).
To assess the significance of both the cAMP-CRP sequences
in rosR expression and their probable responsiveness to the
carbon source, Rt24.2 strain derivatives carrying either i) plas-
mid pEP1 with the entire rosR upstream sequence; ii) plasmid
pEP3A lacking the entire P1 promoter and –35 hexamer of P2
but with the second, more conserved AN-TGTGA-N7-
TCGTA-NT element; iii) plasmid pEP3B lacking the first pen-
tamer of the second cAMP-CRP binding site; and iv) plasmid
pEP3C with promoters and both cAMP-CRP binding sites de-
leted were examined. The strains were grown in M1 minimal
medium supplemented with glycerol or glucose as sole carbon
source and LacZ activity was measured (Fig. 4A). In strains
carrying pEP1 and pEP3A plasmids, 40 and 32% reduction,
respectively, in LacZ activity was observed in the presence of
glucose compared with the activity in the presence of glycerol.
In Rt24.2 derivative carrying pEP3C with both cAMP-CRP
binding sites deleted (but also no promoters present), β-galac-
tosidase activities were very low regardless of the carbon
source (Fig. 4A). These results indicate that rosR expression
depends on the nature of the carbon source and is moderately
affected by glucose. Both P1 and P2 promoters (Fig. 1) and at
least the downstream putative cAMP-CRP binding site are
essential for the transcriptional activation of rosR (Fig. 4A).
To further examine probable cAMP-CRP-mediated activa-
tion of rosR transcription, pEP constructs bearing the truncated
promoter region fused to promoterless lacZ were transferred
into E. coli cyaA mutant (VH1000cyaA) and a wild-type
strain. E. coli strains harboring mutations of the cya gene (en-
coding adenylate cyclase) do not synthesize cAMP and fail to
respond to catabolite repression by glucose. The E. coli deriva-
tives were grown in the presence of glycerol or glucose and
LacZ activities were measured (Fig. 4B and C). We observed
that rosR expression was moderately reduced in the presence
of glucose in an E. coli wild-type strain carrying pEP1 to
pEP3A plasmids (Fig. 4B). The level of repression did not ex-
ceed 50% in the wild-type strain. In the case of pEP3B fusion
lacking the first pentamer, the responsiveness of rosR tran-
scription to glucose disappeared (Fig. 4B).
In the E. coli cyaA mutant carrying the same fusions as the
rosR upstream region, LacZ activity was practically invariable,
regardless of the carbon source (Fig. 4C), indicating that
cAMP is important for the responsiveness of rosR transcription
to glucose. Moreover, the level of LacZ activity in the E. coli
cyaA mutant bearing fusions pEP1 to pEP3A was lower (ap-
proximately 30 to 35%) when grown in the presence of glycerol
compared with the E. coli wild-type strain. This suggested
positive regulation of rosR by cAMP-CRP.
Inverted repeats IR1
(AAACTGAGG) and two directly repeated short CAAGT se-
quences that might function as potential regulator binding sites
also were detected in the rosR upstream region (Fig. 1).
The study of the rosR upstream region was supplemented by
analysis of β-galactosidase activity in E. coli and Rt24.2
strains bearing transcriptional fusions of the 3′-end deleted
promoter region with promoterless lacZ gene (Fig. 3). In both
backgrounds, deletion of the –35 to –10 consensus of P1 and
P2 elements (pEP12) resulted in total lack of LacZ activity.
The highest LacZ activity was observed in strain Rt24.2 carry-
ing pEP11 plasmid that contained the entire P1 promoter but
was lacking the cAMP-CRP downstream element. LacZ activ-
ity of pEP11 was threefold higher compared with the pEP10
plasmid additionally bearing the P2 promoter and cAMP-CRP
binding site. In pEP9, increase in LacZ activity (by a factor of
1.58) was observed in comparison with pEP10, indicating
stimulatory effect of the IR1 and IR2 inverted repeats on rosR
transcription. A low level of LacZ activity in pEP8 might be
explained by the presence of the Ros-box bound by RosR pro-
tein, which would result in decreased rosR transcription. This
indicated rosR autoregulation by its own protein product. It
also is possible that the decrease in rosR transcription in pEP8
versus pEP9, in both genetic backgrounds, was caused by inter-
action of global regulatory LysR-type proteins with the LysR
motif, synergistically with the effect of autoregulation (Fig. 3).
In conclusion, transcription of the rosR gene in strain Rt24.2
is directed from the P1 and P2 promoters. In the presence of
glucose, transcriptional activity of rosR is reduced, indicating
(CCTCAGTTT) and IR2
Fig. 2. Comparison of sequences that show similarity to Rhizobium leguminosarum bv. trifolii RosR binding site in the rosR upstream region. Nucleotides
conserved in all sequences are shaded. Arrows indicate inverted repeats.
Vol. 20, No. 7, 2007 / 871
rosR regulation by catabolic repression. Moreover, additional
regulatory elements, first and foremost the RosR-box, modu-
late rosR transcription.
Mapping of the start site of rosR transcription
by primer extension analysis.
To determine the transcription start site of rosR, fluorescent
primer extension analysis was carried out with a 5′-FAM-
labeled oligonucleotide complementary to the 5′ end of the
rosR gene. Two primer extension products displaying similar
abundance were detected (Fig. 5). One of them was 298 bp in
length and the first nucleotide to be transcribed was an adenine
(A) located –273 nucleotides (nt) upstream of the proposed
rosR translation start codon and 8 nt downstream of –10
(TATTTG) sequence of the P1 promoter (Fig 1). The second
primer extension product was 265 bp in length and the first
transcribed nucleotide was a guanine (G) that was located –240
nt upstream of the rosR translation start codon and 6 nt down-
stream of the –10 (TACAAT) sequence of the predicted P2
promoter. Moderate LacZ activity in pEP3, pEP3A, and
pEP3B fusions and complete lack of LacZ activity in the
pEP3C fusion with both promoters deleted confirms the tran-
scriptional activity of the P2 promoter and the presence of the
second –240-nt transcription start site (Figs. 1 and 3).
Overproduction of RosR protein in E. coli.
To overproduce Rt24.2 RosR as a recombinant N-terminally
His-tagged protein, plasmid pQE450 was used (details below).
A substantial portion of the (His)6RosR protein was produced
in an insoluble aggregated form. The soluble fraction of the
protein was subjected to purification by Ni-NTA affinity chro-
matography. We have not observed the deleterious effect of
(His)6RosR overproduction on E. coli growth described in the
case of MucR (Bertram-Drogatz et al. 1997) and Ros protein
Fig. 3. A, Physical and genetic map of Rt24.2 region encompassing rosR gene. Abbreviations: B, BamHI; P, PstI; H, HindIII; S, SphI; N, NotI. B, Transcrip-
tional activity of rosR putative promoter assayed in Escherichia coli DH5α and Rt24.2 strains carrying pMP220 plasmid with fragments of the rosR up-
stream region fused with promoterless lacZ gene. Lengths of polymerase chain reaction products in particular plasmids are shown as horizontal lines. E. coli
strains were grown in Luria-Bertani medium; Rt24.2 strains were grown in 79CA medium with mannitol as carbon source. β-Galactosidase activities for the
pEP0 to pEP12 plasmid fusions are given as the averages from at least three or four independent experiments.
872 / Molecular Plant-Microbe Interactions
(Cooley et al. 1991). Purified (His)6RosR was subjected to so-
dium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and a major band of approximately 18 kDa was de-
tected, in accordance with the deduced molecular weight of
15.7 kDa for the rosR gene product and accounting for the
presence of the N-terminal His6-tag (Fig. 6A). The identity of
(His)6RosR was confirmed by Western blotting with anti-His5-
tag antibodies (Fig. 6B). In the SDS-PAGE analysis of RosR
protein, in addition to a major 18-kDa band, a band of approxi-
mately 38 kDa was detected.
Considering the possibility of self-association of RosR, in
vitro chemical cross-linking with glutaraldehyde was performed.
After treating RosR with even low (0.01%) concentrations of
glutaraldehyde, dimeric and trimeric forms were detected by
Western blotting with anti-His5 antibodies (Fig. 6C). In the
presence of an increasing concentration of glutaraldehyde,
increased amounts of multimeric forms of RosR appeared at
the expense of the monomeric one (Fig. 6C). This implies that
RosR protein could function at least as a dimer.
Binding of (His)6Ros protein to the RosR-box.
We performed EMSA to study DNA binding of purified
(His)6RosR fusion protein. For these experiments, we used a
PCR-amplified 185-bp digoxigenin (DIG)-labeled DNA frag-
Fig. 4. Effect of carbon source on transcriptional activity of the upstream region fragments of the rosR gene assayed in A, Rt24.2 and in B, Escherichia coli wild-
type and C, cyaA mutant strains. Strains were grown in M1 medium supplemented with Tc and 1% glucose or 1% glycerol for 18 h, and β-galactosidase
activities were measured. Data shown are the mean of ± standard deviation of four replicates.
Vol. 20, No. 7, 2007 / 873
ment I (encompassing the region from –193 to –9 nt), match-
ing the predicted RosR-box site in the rosR upstream region
and increasing concentration of the purified (His)6RosR (Fig.
7A). The observed retardation was dependent on protein con-
centration with an increasing amount of protein, the molecular
weight of the DNA-(His)6RosR complex became visibly larger
(Fig. 7A). However, (His)6RosR at 2 ng μl–1 was sufficient to
shift both the total amount of DNA fragment I and the shorter
DNA 78-bp fragment II (region from –158 to –81 nt) present
in the samples (Fig. 7B).
To examine the DNA binding specificity of (His)6RosR,
electrophoretic mobility shift competition assays with unla-
beled oligonucleotide competitor DNA fragments I to IV were
carried out (Fig. 8). A DIG-labeled DNA RosR-box containing
fragment I was used as a target for (His)6RosR and oligonu-
cleotides I to IV as competitors in all reactions (Fig. 8A
through D). DNA fragment I (185 bp) had to be in a 100-fold
excess over labeled DNA in order to effect full competition, as
may be seen by the absence of a visibly shifted band (Fig. 8A).
The shorter fragment II (78 bp), comprising nucleotides –158
to –81 upstream of rosR, was more effective in competition,
because a 10-fold excess of DNA was sufficient for full
competition (Fig. 8B). This probably might be explained by a
higher diffusion rate of smaller fragments (Bertram-Drogatz et
al. 1997). When DNA fragment III (160 bp) containing the
Ros-box of A. tumefaciens was used as a competitor, it had to
be in a 500-fold excess to sufficiently compete with DNA
fragment I (Fig. 8C). Furthermore, DNA fragment IV (158 bp)
containing the MucR-box even in 500-fold excess did not
compete with labeled DNA (Fig. 8D). These data indicated
high specificity of (His)6RosR binding to the RosR-box in
fragment II and lower specificity binding with fragment I. In
competition assays with heterologous fragments III and IV, the
binding was less specific.
rosR mutant and complementation analysis.
To assess the function of RosR in R. leguminosarum bv. tri-
folii and its possible engagement in EPS biosynthesis regula-
tion, as was done in the case of Ros/MucR proteins (Bertram-
Drogatz et al. 1997; Cooley et. al. 1991; Keller et al. 1995), we
have attempted to construct a rosR deletion mutant lacking the
entire ORF. A construct in which the rosR gene was replaced
by a spectinomycin cassette was obtained and introduced into
the Rt24.2 wild-type strain in order to generate the null muta-
tion. However, we were not able to select desirable transconju-
gants. Thus, we subcloned the rosR ORF from pB31 plasmid
into suicide vector pK19mobGII and the construct was used to
transform E. coli S17-1 (discussed below). Verification of in-
sert correctness of several recombinant plasmids by sequence
analyses revealed that the rosR ORF of one of the plasmids,
named pEPB12, had one nucleotide deletion (ΔC in position
+177 nt) (Fig. 1). The deletion caused a frame shift mutation
starting with the 60th amino acid residue and created a nonsense
codon (TGA) in position +277, which resulted in synthesis of
a shorter putative RosR protein (92 instead of 143 aa). In addi-
tion, we identified nucleotide substitutions resulting in L38P
and D41E mutations of RosR. We used pEPB12 plasmid to
construct the rosR mutant by homologous recombination.
pEPB12 was transferred from E. coli S17-1 to R. leguminosa-
rum bv. trifolii 24.2 by conjugation and the clone, named
Rt2440, with altered, dry, wrinkled colony morphology was
isolated (Fig. 9A). The Rt2440 mutant strain induced nodules
on clover but inoculated plants appeared yellowish, indicating
inefficient symbiosis (Table 1). The amount of EPS produced
by the Rt2440 mutant was 35% of wild-type strain Rt24.2.
Symbiotic and EPS deficiency defects in the Rt2440 mutant
were complemented by the wild-type rosR allele (Table 1).
The function of Rt24.2 RosR also was studied by comple-
mentation of rosR mutation in the closely related species R.
etli CE3ΔrosR. The mutant forms very characteristic domed
colonies on agar plates because of their highly hydrophobic
surface, whereas the parental strain CE3 produces flat colonies
with hydrophilic cell surface (Bittinger et al. 1997). To study
the possibility of restoring wild-type morphology of the
CE3ΔrosR mutant by rosR of Rt24.2, plasmid pRC24 was
introduced by conjugation. As a result, flat, mucoid wild-type
colony morphology was restored in mutant CE3ΔrosR (Fig.
9B). EPS production was measured in the exconjugants
CE3ΔrosR(pRC24) and compared with the level of EPS pro-
duced by mutant CE3ΔrosR and wild-type CE3 strains. Nearly
two times more EPS was produced in CE3ΔrosR(pRC24) (glu-
cose equivalents at 1.55 μg/μg of protein) than in wild-type
strain CE3 (glucose at 0.93 μg/μg of protein) and three times
more than in the mutant strain CE3ΔrosR (glucose equivalents
at 0.54 μg/μg of protein).
Complementation tests in which plasmid pRC24 was conju-
gally transferred into the mucR mutant of S. meliloti (Keller et
Table 1. Complementation of the Rhizobium leguminosarum bv. trifolii 2440 rosR mutant by pRC24 carrying the rosR genea
Nodule no. per plant (days after infection)
Strain, plasmid 5 15 21 30 35 Shoot wt (mg plant–1)b EPS (mg mg–1)c
45.8 ± 11.2 (62.7%)
66.5 ± 12.8 (91%)
73.0 ± 14 (100%)
30.2 ± 7.5 (41.4%)
0.34 ± 0.03 (35%)
1.15 ± 0.08 (121%)
0.95 ± 0.07 (100%)
a Given values (± standard deviation) are averages of two independent experiments with 30 plants for each treatment.
b Shoot fresh weight.
c Exopolysaccharide (EPS) production (Glc equivalents in mg/mg–1 of protein).
Fig. 5. Primer extension analysis of rosR promoter. Fluorogram obtained
from a primer extension assay with 20 µg of total RNA and a FAM-labeled
primer corresponding to position +25 to –2 bp of the rosR sequence.
Length of cDNA primer extension products is given above the peaks. Two
high peaks are the GeneScan-500 ROX internal lane standards with the
size of 247 and 300 bases.
874 / Molecular Plant-Microbe Interactions
al. 1995) or ros mutant of A. tumefaciens (Cooley et al. 1991)
did not yield positive results, signifying that the rosR gene of
R. leguminosarum bv. trifolii 24.2 did not complement mucR
or ros mutations in the respective mutant strains.
To assess the effect of multiple copies of rosR on EPS pro-
duction and symbiosis with clover, plasmid pBR24 was conju-
gally transferred into strain Rt24.2. The exconjugants Rt24.2
(pBR24) produced increased amount of EPS (128% of the
wild-type Rt24.2 strain) and induced more nodules on clover
(data not shown). These results suggested that RosR positively
regulates EPS synthesis in R. leguminosarum bv. trifolii 24.2.
Regulatory function of RosR.
To study regulation of rosR transcription, we introduced
pEP1 fusion, where expression of a promoterless lacZ gene
was under the control of the entire upstream region of rosR
into wild-type Rt24.2 and Rt2440 mutant strains (Table 2). Ac-
tivity of β-galactosidase was twofold higher in the Rt2440
rosR mutant than in the wild-type strain, which indicated auto-
regulation of rosR. In the wild-type background, LacZ activity
was decreased in the presence of both the pEP1 fusion and
pBR24 with the rosR gene, confirming the repressive effect of
RosR on its own transcription.
The effect of RosR on transcription of the pssA gene encod-
ing the first IP-glucosyltransferase in the pathway of R. legu-
minosarum EPS repeating unit synthesis (Pollock et al. 1998)
was examined. In the upstream region of the pssA gene of
Table 2. Analysis of regulatory function of the rosR gene
Strain and plasmids
β β-Galactosidase activity
2,896 ± 113
5,854 ± 241
1,917 ± 102
273 ± 20
164 ± 14
408 ± 32
Rt24.2 (pEP1, pBR24)
Rt24.2 (pSF2, pBR24)
aGiven values (± standard deviation) are averages of three independent
Fig. 6. A, Purification of (His)6RosR protein overproduced in Escherichia coli M15 harboring pQE450. Soluble proteins were separated by 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Lane 1, proteins from uninduced E. coli M15(pQE450) cells;
lane 2, proteins after induction with 1 mM IPTG; lane 3, purified (His)6RosR protein eluted from Ni-NTA agarose, and lane 4, proteins molecular mass
standards (kDa). B, Western-blotting analysis with anti-His5 antibodies of soluble proteins from E. coli M15(pQE450) producing (His)6RosR, after 3 h of
induction with 1 mM IPTG, lane 2, (His)6RosR; and lane 1, purified (His)6RosR. Positions monomers and dimers are indicated. C, Western-blotting analysis
of purified (His)6RosR protein with anti-His5 antibodies after treatment with increasing concentration of glutaraldehyde. Positions of monomers, dimers,
trimers, and multimers are indicated. Molecular weight standards (kDa) are indicated at the right.
Vol. 20, No. 7, 2007 / 875
strain Rt24.2, an RosR-binding site putatively able to bind
RosR was identified earlier (Janczarek and Skorupska 2004).
Plasmid pSF2 containing a transcriptional fusion of the pssA
upstream region with promoterless lacZ gene was introduced
into Rt24.2 and the Rt2440 rosR mutant. Absence of RosR
activity in the mutant background resulted in a decrease of β-
galactosidase activity (by a factor of 0.6), indicating an apparent
positive effect of the rosR gene product on pssA transcription
(Table 2). In the wild-type strain, in the presence of additional
copies of rosR (plasmid pBR24), the level of pssA transcrip-
tion was increased by a factor of 1.5. Overall, rosR gene tran-
scription was negatively autoregulated by RosR but the pssA
gene encoding a key enzyme in EPS synthesis was positively
In the present study, the R. leguminosarum bv. trifolii 24.2
rosR gene was identified and overexpressed in E. coli as an N-
terminally His6-tagged fusion protein containing a C-terminal
conserved zinc-finger DNA-binding domain, as predicted in
silico. The Ros/MucR subfamily of C2H2 zinc-finger proteins
comprises global positive or negative regulators of transcrip-
tion of genes involved in diverse functions in prokaryotes, one
being the regulation of EPS synthesis in S. meliloti, A. tumefa-
ciens, and A. radiobacter (Bittinger and Handelsman 2000,
Chou et al. 1998; D’Souza-Ault et al. 1993, Rüberg et al.
1999). RosR of Rt24.2 is very conservative in its amino acid
composition and the C2H2 zinc-finger motif. Transcriptional
regulators that bind DNA usually act as dimers, and the pres-
ence of RosR of Rt24.2 as dimeric and multimeric structures
was demonstrated in this study. Binding of the (His)6RosR
protein to the RosR-box was evidenced by retardation of 185-
and 78-bp DNA fragments, both matching a 22-bp-long con-
sensus sequence. The specificity of (His)6RosR binding to
fragments I and II was demonstrated by competition experi-
ments. Fragment III containing the Ros-box of A. tumefaciens
(Cooley et al. 1991; D’Souza-Ault et al. 1993) competed with
low affinity, because a 500-fold excess of the competitor over
fragment I was needed to achieve competition. The RosR-box
sequence of Rt24.2 is identical to the RosR-box of R. legumi-
nosarum bv. viciae, almost identical to the Ros-box of R. etli,
and similar to the RosR-box of A. radiobacter and A. tumefa-
ciens. The sequence of the MucR-box of S. meliloti is most
divergent (Bertram-Drogatz et al. 1998; Keller et al. 1995) and
MucR protein did not bind the Ros-box of A. tumefaciens,
indicating a high-specificity interaction between MucR and its
DNA target (Bertram-Drogatz et al. 1997).
In addition to the RosR binding sequence, several regulatory
sequences have been mapped in the upstream region of Rt24.2
rosR, indicating a complex transcriptional control of rosR. A
deletion analysis of the region upstream of rosR revealed a de-
pendence of rosR transcription on P1 and P2 promoters con-
taining conserved σ70–dependent –35 or –10 motifs. Primer
extension mapping detected two transcription start sites. Based
on the primer extension data and sequence analysis of the
upstream promoter region, separate –35 and –10 consensus se-
quences could be assigned for each transcript. Based on analy-
ses of the LacZ activities, one could conclude that the P1 pro-
moter is stronger than the P2 promoter, but an insignificant
difference in the amount of both cDNA fragments in the
primer extension assay indicated a comparable level of the two
transcripts. Further experiments are needed to elucidate the
activity of these promoters under different conditions.
These promoter sequences also were active in E. coli strains,
where we observed a two-fold higher LacZ activity of plasmid
fusions compared with native Rt24.2 background, indirectly
indicating autoregulation of the rosR gene. Negative autoregula-
tion of the rosR transcription also was evidenced by increased
rosR expression in the absence of RosR in the Rt2440 mutant
background. The rosR expression was visibly depressed in the
presence of additional copies of rosR. Similarly, negative auto-
regulation of the ros gene in A. tumefaciens (Cooley et al. 1991)
and mucR in S. meliloti (Keller et al. 1995) has been reported.
Plasmid fusions carrying 5′- and 3′-end deletion fragments of
the rosR upstream region with the reporter lacZ gene allowed us
to examine the significance of cAMP-CRP binding sites in acti-
vation of rosR transcription. The 5′-end deletions of both pro-
moters reduced transcription to the background level in both
Rt24.2 and E. coli. Transcription assays of rosR in the E. coli
mutant lacking cyaA, the gene encoding adenylate cyclase,
showed that, in the absence of cAMP, the rosR promoter ceased
Fig. 7. A, Electrophoretic mobility shift analysis with purified (His)6RosR of increasing concentration. Digoxigenin-labeled DNA A, fragment I and B, fragments
I and II matching a consensus RosR-box were added to all reactions to give a final concentration of 0.1 ng µl–1. Protein concentration in B was 2 ng µl–1.
876 / Molecular Plant-Microbe Interactions
to be responsive to glucose. We conclude that cAMP-activated
CRP protein is able to recognize specific DNA sites and posi-
tively regulates rosR expression. Unexpectedly, a 3′-end deletion
construct containing both the promoters and cAMP-CRP bind-
ing site (pEP10 fusion) showed lower transcription than pEP11
fusion. This could be explained by the fact that bacteria were
cultured in complete medium and, under these conditions, the
cAMP-CRP complex probably was not formed. This effect was
not observed in E. coli background where LacZ activity in-
creased with increasing length of the upstream region.
Computer search for cAMP-CRP binding sites in the rosR
promoter region of R. etli revealed a sequence identical to the
one found in R. leguminosarum bv. trifolii 24.2, located at the
same distance (–244 nt) from the RosR start codon. The cAMP-
CRP binding site located upstream of P1 promoter also was
nearly identical (two substitutions). In the cases of S. meliloti
and A. radiobacter, conservative cAMP-CRP binding sites (Sm:
GT-TGTGA-N6-TCCGT-TG; Ar: CG-TGTCA-N6-TCACT-AA)
downstream of the main promoter were found to be highly
similar to E. coli consensus sequence AAN-TGTGA-N6-TCA
CA-T. The cAMP-CRP binding sites located upstream from
the main promoter were much more divergent in comparison
Formation of the cAMP-CRP complex in E. coli and related
bacteria is a prerequisite to transcriptional activation of cata-
bolic genes. Specific binding sites for the complex usually are
located upstream of these promoters; however, CRP-dependent
promoters (class III promoters) regulated by multiple sites or
by CRP in combination with one or more additional protein
factors also have been described (Harman 2001). Although the
mechanism of cAMP-CRP complex function in rhizobia re-
mains unknown, multiple cya genes encoding adenyl cyclases
have been identified and some of them have been cloned and
characterized (Sharypowa et al. 1999; Tellez-Sosa et al. 2002).
In addition, a hypothetical ORF, Crp1 (accession number
DQ384109), with homology to the effector domain of the CAP
family of transcription factors, was identified recently in the
genome of R. leguminosarum bv. trifolii TA1.
Because RosR is considered to be an activator of acidic EPS
biosynthesis in R. leguminosarum bv. trifolii and its intracellular
concentration might be regulated by glucose, a possibility exists
of a regulatory linkage between carbon metabolism and EPS
production. There are several lines of evidence indicating the
dependence of EPS production in R. leguminosarum bv. trifolii
on the identity of the carbon source (Zevenhuizen 1986). Carbon
source-dependent regulation of expression of pss genes in R.
leguminosarum bv. trifolii could be directed by the rosR gene
product that might function as a positive transcriptional regula-
tor of the pssA gene. Possible regulatory effect of RosR protein
on other pss genes remains to be elucidated.
In addition to the regulatory sites discussed above, several
other elements putatively involved in rosR regulation were iden-
tified upstream of rosR, among them a three-times-repeated
LysR-binding motif. LysR-type transcriptional regulators con-
taining helix-turn-helix DNA-binding domain, such as SyrM
protein, are involved in regulation of EPS production in S.
meliloti (Barnett and Long 1997, Dusha et al. 1999). Under ni-
trogen limitation, NtrC and SyrM proteins act as positive regu-
lators of succinoglycan production. The significance of LysR
elements in rosR expression and of other, long and short in-
verted repeats remains to be elucidated. In conclusion, expres-
sion of the rosR gene encoding a transcriptional regulator re-
quires complex control on the transcriptional level, possibly
involving interaction of different proteins.
Numerous attempts to mutate the rosR gene in strain Rt24.2
eventually resulted in the construction of the Rt2440 mutant,
which formed colonies with characteristic wrinkled, dry mor-
phology and produced less EPS compared with the wild-type
strain. The phenotype of this mutant and the fact that additional
copies of rosR in strain Rt24.2(pBR24) caused an increase in the
amount of EPS in culture supernatant indirectly confirmed posi-
tive regulation of EPS synthesis by RosR. The Rt24.2(pBR24)
strain induced more pink nodules on clover roots than the paren-
tal strain and the weight of green part of plants was significantly
higher compared with control plants (data not shown). A similar
phenomenon was observed in the case of R. etli ΔrosR comple-
mented by the rosR gene, which was more competitive than the
parental strain (Bittinger et al. 1997). These data indicate a pos-
sible transcriptional activation of several symbiosis-related
genes by the overproduced RosR protein.
Fig. 8. Binding specificity of purified (His)6RosR protein with RosR-box
studied by electrophoretic mobility shift assays. Digoxigenin (DIG)-
labeled DNA fragment I was added to the reactions to give a final con-
centration of 0.1 ng µl–1. Protein was added in a final concentration 2 ng
µl–1. Unlabeled competitor oligonucleotides (I to IV) were added in in-
creasing amounts from 2- to 500-fold excess over the DIG-labeled DNA
fragment I. A, DNA fragment I (185 bp) and B, DNA fragment II (78 bp)
contain the RosR-box of Rhizobium leguminosarum bv. trifolii, C, DNA
fragment III (160 bp) contains the Ros-box of Agrobacterium tumefaciens,
and D, DNA fragment IV (158 bp) matches the MucR-box of Sinorhizo-
Vol. 20, No. 7, 2007 / 877
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions.
Bacterial strains and plasmids used in the present study are
listed in Table 3. R. leguminosarum, R. etli, and A. tumefaciens
strains were grown in 79CA medium with 1% mannitol or 1%
glycerol as carbon source at 28°C (Vincent 1970). E. coli strains
typically were grown in Luria-Bertani (LB) medium at 37°C
(Sambrook et al. 1989). To study the expression of rosR in the
Rhizobium spp. or E. coli, the strains were cultured for 24 h in
minimal M1 medium (Sambrook et al. 1989) supplemented with
1% mannitol or glucose, and vitamin solution at 2 ml/liter, pre-
pared according to Brown and Dilworth (1975). Antibiotics
were supplemented as required at the following final concentra-
tions: for E. coli, kanamycin at 25 or 40 μg ml–1, ampicillin at
100 μg ml–1, tetracycline at 10 μg ml–1, and gentamicin at 10 μg
ml–1; and for Rhizobium and Agrobacterium spp., streptomycin
at 200 μg ml–1, nalidixic acid at 40 μg ml–1, tetracycline at 10 μg
ml–1, gentamicin at 10 μg ml–1, and kanamycin at 40 μg ml–1.
Complementation tests were carry out by triparental conju-
gation (Figurski and Helinski 1979) using pRC24 plasmid
bearing rosR of Rt24.2 as donor and R. etli CE3ΔrosR, S.
meliloti 3131 mucR, or A. tumefaciens NTR1 rosR mutant
strains as recipients. Exconjugants were selected on 79CA me-
dium supplemented with the appropriate antibiotics.
Standard techniques were used for plasmid isolation, restric-
tion enzyme digestion, ligation, DNA cloning, transformation,
and agarose gel electrophoresis (Sambrook et al. 1989). Se-
quencing was performed using the BigDye terminator cycle
sequencing kit (Applied Biosystems, Foster City, CA, U.S.A.)
and the ABI Prism 310 sequencer. Database searches were
done with the BLAST and FASTA programs available from the
National Center for Biotechnology Information (Bethesda,
MD, U.S.A.) and European Bioinformatic Institute (Hinxton,
U.K.). The putative promoter sequences were predicted using
Neural Network Promoter prediction at the Berkeley Droso-
phila Genome Project.
Cloning of rosR gene.
Genomic fragments of Rt24.2 and RtTA1 containing the
entire rosR ORF were amplified using the following primers:
forward, RosA 5′-GCGGATCCGCGACTTTACCAGATTTA-
3′ for Rt24.2; forward, RosB 5′-GTCACGCTCTTCGGAATT
CAGGGGT-3′ for RtTA1; and reverse, RosD 5′-TCGGATCCT
GTCGGCAAAGCATAAGA-3′ for both Rt24.2 and RtTA1
strains. The introduced BamHI and EcoRI recognition sites are
underlined. The 1,174-bp PCR product for Rt24.2 and 856-bp
fragment for RtTA1 were cloned into the respective sites of
pUC19 vector resulting in pB31 and pB35 plasmid, respec-
tively. The inserts of pB31 and pB35 containing the entire rosR
genes were sequenced. rosR Rt24.2 and RtTA1 sequences are
available in GenBank database under accession numbers
AY683453 and AY683454, respectively.
Construction of plasmids with nested deletions
of the rosR promoter region.
To construct the plasmids with deletions of the rosR pro-
moter region, plasmid pMP220 carrying a promoterless lacZ
gene was used. For each 5′-end deletion fusion, an upstream
primer for the putative promoter region of rosR, with intro-
duced EcoRI restriction site, and the common reverse primer
RR1, which annealed within the coding sequence of rosR with
introduced XbaI restriction site, were used to amplify DNA
fragments from pB31. For construction of 3′-end deletion fu-
sions, common pEP1 primer and a set of reverse (pEP8 to
pEP12) primers were used (Table 4). The PCR products were
digested with EcoRI and XbaI restriction enzymes and inserted
between the EcoRI and XbaI sites of pMP220 plasmid, result-
ing in plasmids pEP0 to pEP12 containing nested deletions of
rosR promoter fused to a promoterless lacZ gene. The con-
structed plasmids were transferred from E. coli DH5α into R.
leguminosarum bv. trifolii 24.2 by triparental conjugations. β-
galactosidase activities were determined by the method of
Miller (1972) and expressed in Miller units. The reported val-
ues are averages of three to six independent measurements.
Mutagenesis of rosR.
The 1,100-bp fragment of pB31 plasmid was PCR amplified
using pEP1 and RosD primers. PCR product was digested with
EcoRI/BamHI enzymes, cloned into the respective sites of the
suicide vector pK19mobGII, and E. coli S17-1 cells were
transformed with this construct. Inserts of several recombinant
plasmids were sequenced and, in the insert of the plasmid
Fig. 9. Complementation of the A, Rhizobium leguminosarum bv. trifolii 2440 and B, R. etli ΔCE3 mutants by pRC24 plasmid containing the rosR gene of
Rt24.2. Colonies were grown for 7 and 12 days on 79CA medium.
878 / Molecular Plant-Microbe Interactions
named pEPB12, one nucleotide deletion (ΔC in position +177
nt) was found which caused a frameshift mutation starting
with the 60th amino acid residue. The nonsense codon (TGA)
in position +277 was created which resulted in synthesis of a
shorter putative RosR protein (92 instead of 143 aa) (Fig. 1).
In addition, nucleotide substitutions resulting in L38P and
D41E mutations of RosR were identified. pEPB12 was trans-
ferred from E. coli S17-1 to R. leguminosarum bv. trifolii 24.2
by conjugation and transconjugants were selected on 79CA
medium with kanamycin and nalidixic acid. We isolated a
clone, named Rt2440, with altered, wrinkled colony morphol-
ogy. Recombination between pEPB12 and the R. leguminosa-
rum bv. trifolii 24.2 rosR gene was verified by PCR amplifica-
tion using pEP1 and RosD primers with Rt2440 as a template
and Pfu polymerase (Roche, Branchburg, NJ, U.S.A.) followed
by sequencing of PCR product. Sequencing of the Rt2440
PCR fragment confirmed the presence of the mutations men-
Construction of rosR containing expression plasmid.
The rosR gene was amplified by PCR from the pB31 plas-
mid as a template using forward primer EK1-5′-CCAGGAG
AAAAAGGATCCCGGATA-3′ with an introduced BamHI
recognition site (underlined) and the reverse primer EK2-5′-
GTCGTCGACGCTTTCGAAAAAGCT-3′ with an introduced
SalI recognition site (underlined) and Ready Mix Taq PCR Re-
action Mix (Sigma-Aldrich, St. Louis). The PCR product was
digested with BamHI and SalI restriction enzymes and inserted
into the corresponding sites in pUC19, yielding the pEK450
plasmid. The sequence of PCR product was verified by se-
Table 3. Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristicsa Sources or reference
Wild type, Rifr, Nxr
Wild type, Rifr, Smr
Rt24.2 derivative carrying rosR with one nucleotide deletion
Spontaneous Smr derivative of CFN42; Smr, Nxr
CE3 with rosR deleted; Smr, Nxr
Wild type, Smr
Rm2011 mucR31::Tn5, Smr, Kmr
Flagellum-free derivative of C58, Smr
NT1REB with ros mutation, Smr
supE44 ΔlacU169 (ϕ80 lacZΔ M15) hsdR17 recA1endA1gyrA96 thi-1 relA1
Lac–, Ara–, Gal–, Mtl–
Derivative of MG1655, lacZ, lacI, pyrE+
Derivative of VH1000, ΔcyaA::Km
Skorupska et al. 1995
Chakravorty et al. 1982
Noel et al. 1984
Bittinger et al. 1997
Casse et al. 1979
Keller et al. 1995
Lai and Kado 1998
Cooley et al. 1991
Sambrook et al. 1989
Gaal et al. 1997
Gaal et al. 1997
Sambrook et al. 1989
Figurski, Helinski 1979
Stanley et al. 1987
Kovach et al. 1995
Katzen et al. 1999
Spaink et al. 1987
Cloning and sequencing vector, Apr
Tra+ helper plasmid, Kmr
IncP, mob, Tra– cosmid vector, Tcr
Mob+, lacZα, Gmr
ori p15A, Kmr, lacIq
Mob, lacZα, gusA, Kmr
ori ColE1, Apr, expression vector
IncP, mob, promoterless lacZ, Tcr
pUC19 with 1,174-bp BamHI fragment containing rosR of Rt24.2
pUC19 with 856-bp EcoRI-BamHI fragment containing rosR of RtTA1
pBBR1MCS-5 with 1,174-bp BamHI fragment containing rosR of Rt24.2
pRK7813 with 1,174-bp BamHI fragment containing rosR of Rt24.2
pK19mobGII with 1,100-bp EcoRI-BamHI fragment containing rosR of 24.2 with one
pMP220 carrying the –450-bp upstream region to +243 bp of the rosR coding region
pMP220 carrying the –403-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –357-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –281-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –263-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –250-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –235-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –219-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –159-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –96-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –41-bp upstream region to +243-bp fragment of rosR coding region
pMP220 carrying the –403-bp to –32 bp of rosR upstream region
pMP220 carrying the –403-bp to –185 bp of rosR upstream region
pMP220 carrying the –403-bp to –232 bp of rosR upstream region
pMP220 carrying the –403-bp to –268 bp of rosR upstream region
pMP220 carrying the –403-bp to –348 bp of rosR upstream region
pMP220 with one 1,084-bp SacI fragment carrying promoter region of pssA
pUC19 with 450-bp BamHI-SalI fragment containing rosR of Rt24.2, Apr
pQE32 with 450-bp BamHI-SalI fragment containing rosR of Rt24.2, Apr
Janczarek and Skorupska 2004
a Rifr, Nxr, Smr, Apr, Tcr, Gmr, and Kmr = resistant to rifampicin, nalidixic acid, streptomycin, ampicillin, tetracycline, gentamicin, and kanamycin,
Vol. 20, No. 7, 2007 / 879
quencing. The insert then was subcloned into the expression
vector pQE-32 (QIAexpress; Qiagen, Hilden, Germany), gen-
erating plasmid pQE450 for the overproduction of a recombi-
nant RosR carrying an N-terminal His6-tag.
Primer extension analysis.
Total RNA was isolated from R. leguminosarum bv. trifolii
24.2 cells using the Trizol reagent (Sigma-Aldrich) according to
the manufacturer’s instructions. RNA was quantitated by deter-
mining the absorbance at an optical density at 260 nm (OD260)
and stored at –80°C. The rosR transcriptional start site was iden-
tified by an automated fluorescent primer extension method as
was described by Lloyd and associates (2005). A 5′-FAM-
labeled oligonucleotide primer (5′-CATTGCCGGTCGCTATAT
CCGTCATA-3′) complementary to a region near the 5′ end of
the rosR gene (from –2 to +25 bp) was used. RNA was treated
with DNase I (Roche) before use. Primer extension reactions
were carried out where FAM-labeled primer at a final concentra-
tion of 10 pmol was added to 20 μg of total RNA in the final
volume of 20 μl. The cDNA synthesis was performed in one
step using AMV RT enzyme (Promega Corp., Madison, WI,
U.S.A.) (Lloyd et al. 2005). Samples of FAM-labeled cDNA
were separated on an ABI PRISM 3100 Genetic Analyzer
(Applied Biosystems) capillary electrophoresis instrument using
techniques and parameters suggested by the manufacturer. The
ROX-500 size standards (Applied Biosystems) were included in
each run to determine fragment lengths.
Overproduction and purification of RosR protein.
E. coli M15 carrying the plasmid pQE450 was grown in 200
ml of LB medium supplemented with ampicillin (100 μg ml–1)
and kanamycin (25 μg ml–1) at 30°C. When the culture had
reached an OD600 of approximately 0.4, the expression of rosR
was induced by addition of isopropyl-β-D-thiogalactopyrano-
side (final concentration 1 mM). The cells were harvested 3 to
4 h postinduction and lysed in lysis buffer (50 mM NaH2PO4,
pH 8.0, 300 mM NaCl, 5 mM imidazole, 1 mM phenylmethyl-
sulfonyl fluoride, and lysozyme at 1 mg ml–1) on ice for 30
min. Following this, the samples were sonicated on ice using
six 10-s bursts at 200 to 300 W with a 10-s cooling period be-
tween each burst. Crude cell lysates were clarified by centrifu-
gation at 10,000 × g for 40 min. The supernatant was applied
to a nikiel-nitrilotriacetic acid resin (Ni-NTA) (Qiagen) and
the (His)6RosR protein was eluted from the column with 200
mM imidazole. Column fractions containing the (His)6RosR
protein were pooled, two times dialyzed against buffer (20
mM Tris-HCl, pH 8.0, 100 mM NaCl, and 10% vol/vol
glycerol) and stored at –20°C. Protein concentration was
determined by the Bradford method using Bio-Rad Protein
Assay (BioRad Laboratories, München, Germany). The
purification yield was approximately 1.5 mg of (His)6RosR
protein per 200 ml of E. coli M15 culture.
For EMSA, DNA fragments of rosR promoter matching the
RosR-box sequence were amplified by PCR using plasmid pB31
as a template. The primers used for PCR amplification are listed
in Table 5. The fragments were labeled at 3′ ends with DIG-11-
ddUTP and terminal transferase using DIG Gel Shift Kit
(Roche), according to the manufacturer’s instruction. Unlabeled
specific competitor fragments were obtained by PCR amplifica-
tion using the same primers. In a routine assay, DIG-labeled
DNA fragments I and II were mixed with purified (His)6RosR in
reaction buffer containing 20 mM Hepes, pH 7.6, 1 mM EDTA,
10 mM (NH4)2SO4, 1 mM dithiothreitol (DTT), 0.2% wt/vol
Tween 20, 30 mM KCl, poly-L-lysine at 5 μg ml–1, and poly[d(I-
C)] at 0.5 μg ml–1 to a total volume of 20 μl. The reaction mix-
ture was incubated for 15 min at 25°C. In competition assays,
(His)6RosR protein was added to DIG-labeled DNA fragments
in the presence of various concentrations of specific competitor
DNA fragments. After incubation, the reaction mixtures were
loaded onto a nondenaturing 5% polyacrylamide gel with 10%
glycerol and resolved in Tris-glycine buffer (25 mM Tris, 190
mM glycine) supplemented with 5 mM MgCl2, 1 mM EDTA,
and 0.1 mM DTT at 7 V/cm for 3 h. DNA protein complexes
were electrotransferred onto a nylon membrane and visualized
by chemiluminescent detection according to the instruction
manual of DIG Gel Shift Kit (Roche).
For chemical cross-linking, 1 μg of RosR protein in 50 mM
potassium phosphate buffer (pH 7.6) was incubated for 15 min
at room temperature with increasing concentration of glutaral-
dehyde (0.01 to 0.25%, vol/vol). The reactions were stopped
by addition of loading buffer, heated (5 min at 95°C), and ana-
lyzed by SDS-PAGE.
Table 5. Primers used for polymerase chain reaction (PCR) of the DNA fragments used in electrophoretic mobility shift assays
Sequence of primers (5′ ′–3′ ′)
Length of PCR
Fragment I with RosR-box of Rt24.2
Fragment II with RosR-box of Rt24.2
Fragment III with Ros-box of Agrobacterium tumefaciens NT1REB
Fragment IV with MucR-box of Sinorhizobium meliloti 2011
Table 4. DNA primers used in the construction of semi-nested deletion of
the rosR upstream region fused with promoterless lacZ in plasmid pMP220a
Name of primer Sequence of primers (5′ ′–3′ ′)
a Sequences for the EcoRI and XbaI restriction sites are underlined.
880 / Molecular Plant-Microbe Interactions
SDS-PAGE-separated proteins were electroblotted onto Im-
mobilon P (polyvinylidene diflouride) membranes (Millipore,
Bedford, MA, U.S.A.), and probed with mouse anti-His5 tag
antibodies (Qiagen), followed by alkaline phosphatase-conju-
gated anti-mouse immunoglobulin antibodies (Sigma-Aldrich).
Bands were visualized with nitroblue tetrazolium chloride/5-
bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich).
For EPS isolation, 5-ml cultures of rhizobia were grown in
79CA medium with 0.5% glycerol for 3 days at 28°C in a ro-
tary shaker. EPS was precipitated from culture supernatants
with 10 volumes of 96% ethanol. The precipitate was collected
by centrifugation, lyophilized, resolved in water, and analyzed
for carbohydrates according to Loewus (1952). Total sugars
content was calculated in glucose equivalents.
Red clover (Trifolium pratense cv. Ulka) seed were surface
sterilized, germinated, and grown as described previously
(Skorupska et al. 1995). The clover plants were inspected for
root nodule formation and, after 5 weeks, plants were har-
vested and green matter production was estimated by weighing
We thank J. Handelsman (University of Wisconsin, Madison, U.S.A.)
for providing the R. etli CE3 and CE3 ΔrosR strains. We are grateful to C.
Kado (Davis Crown Gall Group, University of California, Davis, U.S.A.)
for providing the NT1REB and NTR1 strains of A. tumefaciens, and A.
Becker (Lehrstuhl für Genetic, Bielefeld University, Bielefeld, Germany)
for providing the Rm2011 and Rm3131 strains of S. meliloti. We also
thank R. Gourse (via G. Węgrzyn, Department of Molecular Biology, Uni-
versity of Gdańsk, Poland) for providing the E. coli VH1000 and
VH1000cyaA strains. We thank M. Małek for excellent technical assis-
tance. This work was supported by the Polish Committee for Scientific Re-
search grant number 2 P04A 03426.
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