JOURNAL OF BACTERIOLOGY, Apr. 2003, p.

24022409
0021-9193/03/$08.000 DOI: 10.1128/JB.185.8.24022409.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.

Vol. 185, No. 8

Control of rep Gene Expression in Plasmid pGA1 from

Corynebacterium glutamicum
Tatiana Venkova-Canova, Miroslav Patek, and Jan Nesvera*
Institute of Microbiology, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4,
Czech Republic

The cryptic multicopy plasmid pGA1 (4,826 bp) from Corynebacterium glutamicum LP-6 belongs to the fifth
group of rolling-circle-replicating plasmids. A determinant, which negatively controls pGA1 replication, was
localized in the leader region of the rep gene coding for the initiator of plasmid replication. This region, when
cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1
derivative pKG48. A promoter and a single transcriptional start site were found in the rep leader region in
orientation opposite to the rep gene. These results suggest that a small countertranscribed RNA (ctRNA) (ca.
89 nucleotides in length), which might inhibit translation of pGA1 rep gene, is formed. Analysis of predicted
secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria.
Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which
proved a negative role of the ctRNA in control of pGA1 copy number. A region between the promoters Prep and
P-ctRNA with a potential to form secondary structures on both ctRNA and rep mRNA was found to cause low
activity of the rep promoter even when promoter P-ctRNA was deleted. Thus, the sequence within the rep leader
region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively
influencing expression of the pGA1 rep gene.
ing of a small repressor protein (CopG) to the rep promoter
(7).
The cryptic multicopy plasmid pGA1 (4,826 bp) from
Corynebacterium glutamicum LP-6 (39) replicates by the rolling-circle mechanism. Its minimal replicon (1.7 kb) contains
the rep gene, coding for the Rep protein (22), and the doublestrand origin of replication (1). The deduced amino acid sequence of the pGA1 Rep protein is highly homologous to
those of the Rep proteins of other small cryptic plasmids from
corynebacteria, pSR1 from C. glutamicum (2) and pYM2 from
C. efficiens (GenBank accession no. AB084384). Similarity of
these Rep proteins to the Rep proteins of larger plasmids from
corynebacteria, namely, pNG2 from C. diphtheriae (45), pAG1
from C. glutamicum (41), pTP10 from C. striatum (40), pCG4
from C. glutamicum (GenBank accession no. AF164956),
pTET3 from C. glutamicum (GenBank accession no.
AJ420072), and five plasmids from C. jeikeium (pK43, pCJ84,
pK64, pKW4, and pB85766; GenBank accession numbers
AF364477, AY048596, AY079086, AF401314, and AF486522,
respectively), was also found. Since no significant homology
between these Rep proteins and the Rep proteins of other
RCR plasmids belonging to the four basic families exists, these
Corynebacterium plasmids were proposed to form the fifth
group of RCR replicons (26).
Plasmid pGA1 contains two genes, per and aes, located outside its minimal replicon, whose products influence positively
stable maintenance of the plasmid. The per gene, whose expression was found to be negatively controlled at the transcriptional level by the pGA1 gene product(s), is the main determinant involved in stable maintenance of pGA1. When present
in trans, per stimulates an increase in copy number and in
segregational stability of the low-copy pGA1 derivatives lacking this gene (22). The aes gene was found to code for an

Bacterial plasmids as extrachromosomal, self-replicating

DNA molecules, encode elements controlling their replication.
To define and to maintain their characteristic copy number in
a given host, plasmids replicating in both theta-type and rolling-circle modes use negative regulatory circuits (34). Inhibition of plasmid replication by increasing the dosage of negatively acting elements in trans has been used to identify these
negative regulators of plasmid replication (24).
The replication of rolling-circle-replicating (RCR) plasmids
has been shown to be regulated by negative control of synthesis
or of activity of their replication initiators (Rep proteins),
which are rate limiting for replication. Small (50 to 250 nucleotides [nt]), untranslated, short-lived, and constitutively synthesized, countertranscribed RNAs (ctRNAs) complementary
to the leader sequences of rep mRNAs are the main regulatory
elements involved in negative control of the rep gene expression (18, 35). In plasmids of the pT181 family and in plasmid
p353-2 (pC194 family), one or two ctRNAs cause premature
termination (attenuation) of rep mRNA synthesis (25, 32).
RCR plasmids of the pMV158 family, and also some plasmids
belonging to the pC194 family, use ctRNA regulation at the
translational level mediated by interference of ctRNA with
Rep synthesis by sequestering the ribosome-binding site of rep
mRNA (8, 20). In plasmids of the pMV158 family, still another
mechanism of negative control of rep expression involves bind-

* Corresponding author. Mailing address: Institute of Microbiology,

Academy of Sciences of the Czech Republic, Vden
ska 1083, CZ-14220
Prague 4, Czech Republic. Phone: (420) 241062398. Fax: (420)
241722257. E-mail: nesvera@biomed.cas.cz.
Present address: UNAM-Centro de Investigacion sobre Fijacion
de Nitrogeno, Programa de Evolucion Molecular, Chamilpa, C.P.
62210, Cuernavaca, Mexico.
2402

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Received 15 October 2002/Accepted 24 January 2003

PLASMID pGA1 FROM CORYNEBACTERIUM GLUTAMICUM

VOL. 185, 2003

2403

TABLE 1. Plasmids used in this work

Relevant characteristicsa

Plasmid

pK19
pS19
pGA1

pET2-PEXctRNA
pET2-PctRNA1
pET2-PctRNA2
pET2-PctRNA3
pET2-PctRNA4
pET2-PctRNA5
pET2-PctRNAmut
pET2-Prep1
pET2-Prep2
pET2-Prep3
pET2-Prep4
pET2-Prep4(PctRNAmut)
pEC6
pEC6-LR

33
This work
22, 39
22
This work
22
This work
42
This
This
This
This
This
This
This

work
work
work
work
work
work
work

This
This
This
This
This

work
work
work
work
work

B. J. Eikmanns
This work

Kmr, kanamycin resistance; Smr, streptomycin resistance; Spr, spectinomycin resistance; Cmr, chloramphericol resistance.
Nesvera et al. (21).
c
Eikmanns et al. (10).
b

accessory element involved in the stable maintenance of the

plasmid pGA1 (44).
To find a regulatory element negatively controlling replication of the plasmid pGA1, we analyzed in detail structure and
function of the region upstream of its rep gene. This analysis
proved the negative role of a small ctRNA in control of plasmid pGA1 copy number and the negative effect of a sequence
in the rep leader region with potential to form secondary structures on the activity of the rep promoter.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. Bacterial strains used in
the present study were Escherichia coli DH5 (14) and C. glutamicum R127 (19).
Plasmids used are listed in Table 1. E. coli strains were grown at 37C in
Luria-Bertani medium (36), C. glutamicum strains were grown at 30C in Casitone-yeast extract (CY) medium (29). Selective conditions in the media were
obtained by using antibiotics at the following concentrations: kanamycin, 20
g/ml; streptomycin, 20 g/ml; spectinomycin, 20 g/ml; and chloramphenicol,
10 g/ml (for C. glutamicum) and 20 g/ml (for E. coli).
DNA manipulations and transformation of E. coli and C. glutamicum. Plasmid
DNA from E. coli was isolated by the method of Birnboim and Doly (5). Plasmid
DNA from C. glutamicum was isolated by a modified alkaline extraction procedure with lysozyme (30). DNA fragments were amplified by the PCR technique.
Restriction enzymes and T4 DNA ligase were used as recommended by the
manufacturers (New England Biolabs or Fermentas). The nucleotide sequences
were determined by using automatic sequencer Vistra (Amersham). Transformation of E. coli DH5 was carried out by the method of Hanahan (14), and
electrotransformation of C. glutamicum was done according to the method of
Liebl et al. (19).
Site-directed mutagenesis. Inactivation of the 10 hexamer of promoter PctRNA was performed by site-directed mutagenesis (17). Two complementary
primers, PCTRNAMD (5-AAGAAATGTCCCTGGCCGAT-3) and PC-

TRNAMR (5-ATCGGCCAGGGACATTTCTT-3), covering positions 2236 to

2255 of pGA1 (GenBank accession no. X90817), and two external primers,
pUC/M13 Reverse (Promega) and PGANRUD (5-AGGCGCTCGCGAAATC
TC-3) covering positions 2433 to 2416 of pGA1, were used in PCRs. The
resulting alteration of the 10 hexamer of the promoter P-ctRNA (TAGAGT 3
CAGGGA) changed only the second codon of the rep gene, leading to exchange
of threonine for serine in the Rep protein. To obtain the plasmid
pKG32PctRNAmut, the final PCR product (739 bp), carrying unique PstI and
NruI restriction sites, was used to replace the wild-type PstI-NruI fragment in
plasmid pKG32.
RNA isolation and primer extension analysis. Total RNA from C. glutamicum
R127 containing plasmid pET2-PEXctRNA was isolated according to the
method of Eikmanns et al. (11). Oligonucleotide CM4 (5-GAAATCTCGTCG
AAGCGTCG-3), covering positions 21 to 40 relative to the translational
start site of the cat gene in plasmid pET2, was used as a primer. It was terminally
labeled with [-32P]ATP by using T4 polynucleotide kinase (Amersham). The
primer extension reaction was done by the method of Peters-Wendisch et al.
(31).
Determination of the incompatibility effect caused by a cloned gene. Incompatibility effect of a cloned gene in biplasmid C. glutamicum cells was determined
by monitoring maintenance of the resident plasmid during cultivation under
conditions selective for the plasmid carrying the possible determinant negatively
controlling replication. The overnight cultures were serially diluted (1:200) in
fresh CY medium without antibiotic, and the cells were plated on CY plates
without antibiotic. After growth to single colonies, replica plating on CY plates
containing antibiotics selective for only the tested plasmid (kanamycin) and for
both plasmids (kanamycin and chloramphenicol) was performed. The percentage
of the kanamycin-resistant (Kmr) clones was determined, and the presence of
plasmid DNA was checked in selected isolates.
Determination of plasmid copy number and plasmid maintenance. The number of plasmid copies per C. glutamicum chromosome was determined in total
lysates prepared by the method of Noirot-Gros and Ehrlich (23) from plasmidharboring C. glutamicum cells grown in the presence of kanamycin. The DNA
samples were run on 0.8% agarose gel and then stained with SYBR-Green I

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pKG48
pSG48
pKG32
pK32PctRNAmut
pET2

Kmr; E. coli vector (2.7 kb)

Smr Spr; E. coli vector (3.0 kb) (pK19 derivative, Kmr determinant was
exchanged for Smr Spr determinant from pCG4)b
Cryptic plasmid from C. glutamicum LP-6 (sequence: GenBank/EMBL
accession no. X90817)
pGA1 linearized in EcoRI site (nt 1788) in pK19
pGA1 linearized in EcoRI site (nt 1788) in pS19
BamHI fragment of pGA1 (nt 16074826) in pK19
pKG32 with mutation in the 10 hexamer of promoter P-ctRNA
Kmr; E. coli-C. glutamicum promoter-probe vector (7.5 kb, replicon of
pBL1, promoterless cat gene)
Promoter P-ctRNA (PCR fragment of pGA1, nt 2395 to 2219) in pET2
Promoter P-ctRNA (PCR fragment of pGA1, nt 2395 to 2198) in pET2
Promoter P-ctRNA (PCR fragment of pGA1, nt 2395 to 2172) in pET2
Promoter P-ctRNA (PCR fragment of pGA1, nt 2395 to 2159) in pET2
Promoter P-ctRNA (PCR fragment of pGA1, nt 2395 to 2151) in pET2
Promoter P-ctRNA (PCR fragment of pGA1, nt 2395 to 2147) in pET2
Promoter P-ctRNA with mutated 10 hexamer (PCR fragment of pGA1,
nt 2395 to 2172) in pET2
Promoter Prep (PCR fragment of pGA1, nt 2007 to 2150) in pET2
Promoter Prep (PCR fragment of pGA1, nt 2007 to 2212) in pET2
Promoter Prep (PCR fragment of pGA1, nt 2007 to 2230) in pET2
Promoter Prep (XbaI-NheI fragment of pGA1, nt 1979 to 2275) in pET2
Promoter Prep and promoter P-ctRNA with mutated 10 hexamer (XbaINheI fragment of pGA1, nt 1979 to 2275) in pET2
Cmr; derivative of pEC5c with unique BamHI site
PCR fragment of pGA1 containing rep gene leader region (nt 2151 to 2395)
in pEC6

Source or
reference

2404

VENKOVA-CANOVA ET AL.

J. BACTERIOL.

(Sigma). The electrophoreograms were scanned by Kodak Gel Scanner and

analyzed by the software supplied with the scanner. Estimations of plasmid copy
number per chromosome were done by using the following equation: [(signal of
plasmid CCC OC forms 1.36)/(signal of chromosome)] [(size of C.
glutamicum chromosome/plasmid size)], where CCC is the covalently closedcircle form of the plasmid and OC is the open-circle form of the plasmid. A value
of 3,309 kb (GenBank accession no. NC003450) was used as the size of C.
glutamicum chromosome.
For determination of plasmid maintenance, plasmid-harboring C. glutamicum
strains grown overnight were diluted (1:200) in fresh CY medium and, after
serial dilutions, the cells were plated on CY medium without antibiotics and then
replica plated on CY plates containing kanamycin. The percentage of Kmr clones
was determined, and the presence of plasmid DNA was checked in selected
isolates.
Enzyme assay and determination of MIC. The specific activity of chloramphenicol acetyltransferase (CAT) in C. glutamicum cell extracts was determined
by the method of Shaw (38). The MIC of chloramphenicol in E. coli and C.
glutamicum strains was determined on Luria-Bertani and CY plates, respectively,
by the method of Ozaki et al. (27).
Calculation of theoretical efficiency of transcription termination. Free energy
(G) of formation of hairpin RNA structures (computer predicted by using the
DNAstar-GeneQuest program) was calculated according to the method of Freier
et al. (13). The theoretical efficiency of termination was calculated by using the
algorithm developed by dAubenton-Carafa et al. (6).

RESULTS
A determinant negatively controlling plasmid pGA1 replication is located upstream of the rep gene. Determinants negatively controlling replication of RCR plasmids are predominantly located upstream of the rep genes (18). To find such
determinant(s) on plasmid pGA1, the fragment of pGA1 DNA
(nt 2151 to 2395 in Fig. 1) covering a substantial part (90 of 93
nt) of the rep gene leader region, was cloned into the multicopy
vector pEC6, a derivative of pBL1 (37) compatible with pGA1.
A sequence with potential to generate a secondary structure

that may serve as a rho-independent transcriptional terminator

in a direction opposite to the rep gene is present in the rep
leader region (structure IR1 in Fig. 1). The resulting construct
pEC6-LR was introduced into C. glutamicum R127 harboring
plasmid pKG48 (which contains the complete pGA1 sequence), and maintenance of pKG48 was monitored. After 25
generations of growth under conditions selective for pEC6-LR,
only 58% of the C. glutamicum cells containing pEC6-LR harbored the plasmid pKG48 also. On the other hand, 100% of
the C. glutamicum cells containing vector pEC6 harbored also
pKG48 under the same conditions. This incompatibility effect
indicates that the cloned DNA fragment carries a trans-acting
determinant negatively controlling plasmid pGA1 replication.
A promoter is present in the rep leader region in orientation
opposite to the rep gene. Plasmid pGA1 fragment (nt 2198 to
2395 in Fig. 1) containing part of the rep leader region but
lacking the sequence IR1, which resembles a rho-independent
terminator (Fig. 1), was cloned into the promoter-probe vector
pET2 in orientation opposite to that of the rep gene and tested
for promoter activity in C. glutamicum cells. This fragment
carried by the resulting plasmid pET2-PctRNA1 exhibited high
transcriptional activity. The level of chloramphenicol resistance (MIC) was found to be 90 g/ml. The corresponding
CAT activity in cell extract was 0.462 0.010 mol min1
mg of protein1. When the vector pET2 was used as a control,
the MIC and CAT activity values were 5 g/ml and 0.001
mol min1 mg of protein1, respectively. To map the 5
end of the transcript pertinent to this newly discovered promoter, primer extension analysis of total RNA isolated from
C. glutamicum R127 harboring plasmid pET2-PEXctRNA (carrying fragment nt 2219 to 2395) was performed. A single band

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FIG. 1. Part of the pGA1 sequence relevant to the data presented (the coordinates used are those from the GenBank/EMBL pGA1 sequence,
accession number X90817). The DNA sequence of the rep gene leader region (LR) is marked in boldface; 1 indicates the first base of the
transcript, which is shown as a boldface underlined letter; and the putative 10 and 35 hexamers of the promoters are underlined. IR, inverted
repeat; SD, putative Shine-Dalgarno site whose sequence is underlined. The start codon of rep gene is underlined; the first 9 amino acids of the
Rep protein are shown; and the enzyme sites are marked in italics. E1, E2, E3, E4, and E5 indicate the positions of the 3 ends of the fragments
cloned into the vector pET2 (see the resulting plasmids pET2-PctRNA1, pET2-PctRNA2, pET2-PctRNA3, pET2-PctRNA4, and pET2-PctRNA5
in Table 3).

VOL. 185, 2003

PLASMID pGA1 FROM CORYNEBACTERIUM GLUTAMICUM

2405

TABLE 2. Number of copies per chromosome of C. glutamicum

and maintenance of the derivatives of plasmid pGA1
in C. glutamicum

Derivative of pGA1

pKG48
pKG32
pKG32ctRNAmut
b

FIG. 2. Transcriptional start site respective to the promoter

P-ctRNA as determined by primer extension. Letters above represent
the products of the DNA sequencing (A, C, G, and T represent the
dideoxynucleoside triphosphates with which the sequencing was done).
P indicates the product of primer extension; the DNA sequence read
from the gel and its complementary sequence are shown on the right
side. The respective transcriptional start site is shown in boldface ().
The 10 hexamer is also indicated.

was detected as a result of reverse transcription with oligonucleotide primer complementary to the vector pET2 (Fig. 2).
The transcriptional start site, mapped as base G in Fig. 2, is
located 6 bp upstream of the translational start site of the
pGA1 rep gene (Fig. 1). The putative promoter hexamers
(10, TAGAGT; 35, GTGACT) (Fig. 1 and 2) show similarity to those of the C. glutamicum promoter consensus sequence (TANAAT and TTGGCA, respectively [28, 43]). On
the basis of these results, we conclude that pGA1 encodes a
small countertranscribed RNA (ctRNA), complementary to
the leader region of the rep mRNA, which can be involved in
negative control of expression of the pGA1 rep gene. The
newly discovered promoter, designated P-ctRNA, was found to
be constitutive, since no negative effect of presence of plasmid
pSG48, containing the complete pGA1 sequence, on its activity
was observed. (CAT activity in cell extract of C. glutamicum
harboring pET2-PctRNA1 plus pSG48 was 0.51 0.05
mol min1 mg of protein1.)
Effect of inactivation of the promoter P-ctRNA on plasmid
copy number. To test the effect of putative ctRNA on pGA1
copy number, inactivation of promoter P-ctRNA, abolishing
synthesis of ctRNA, was performed. Its 10 hexamer TAGAGT was altered by site-directed mutagenesis to the sequence CAGGGA. The fragment containing the mutated 10
hexamer was cloned into the promoter-probe vector pET2. No
promoter activity of this fragment was observed in cell extracts
of C. glutamicum harboring the resulting plasmid pET2-PctRNAmut, which confirmed the essential role of the 10 hexamer for transcriptional activity (the MIC of chloramphenicol
was 5 g/ml, and the CAT activity was 0.01 mol min1
mg of protein1). To test the phenotypic effect of this mutation, the region carrying wild-type P-ctRNA was exchanged for

ctRNA

per
gene

Mean copy no.a

SD

Maintenance
after 25
generations
(%)b

37.1 7.1
6.3 3.5
49.7 9.5

100
0.2
100

Values represent the means from at least four independent experiments.

Expressed as the percentage of Kmr clones.

that carrying its mutated 10 hexamer in the low-copy-number

and segregationally unstable derivative of pGA1, plasmid
pKG32. As shown in Table 2, inactivation of promoter PctRNA caused approximately eightfold increase in the number
of copies of the plasmid pKG32PctRNAmut in comparison
with that of the original plasmid pKG32. The observed segregational stability of pKG32PctRNAmut, differing from extremely unstable maintenance of pKG32, is most probably a
consequence of the increased number of plasmid copies. These
results proved the negative role of the ctRNA in control of
pGA1 copy number in C. glutamicum cell.
Analysis of the predicted secondary structure of the pGA1encoded ctRNA and determining its length. Computer-predicted secondary structure of the pGA1-encoded ctRNA
shows the formation of two hairpin structures (Fig. 3A). The
first hairpin (S1), which would start 4 nt downstream of the
transcriptional start site of the ctRNA, possesses a 10-bp GCrich stem with a small internal loop (G 11.5 kcal/mol).
The sequence of the 5 end of the stem is complementary to
the putative Shine-Dalgarno sequence of the rep gene. The
sequence at the loop of the predicted secondary structure
(5-CUGAUGA-3) exhibits the U-turn motif (12), which fits
the consensus sequence of U-turns in prokaryotic antisense
RNAs or in their target RNAs (5-YUNR-3, where Y
pyrimidine, U uracil, N any base with preference of purine
[most often G], and R purine) (Fig. 3B). The U-turn motif
is considered to promote RNA-RNA pairing (12). The second
possible structure (T) might be formed 2 bp downstream of the
first secondary structure. This hairpin consists of a 14-bp GCrich stem and a 4-nt loop. Since five U residues are present 5
nt (instead of the usual 3 nt) downstream of its 3 end, we
assume that this hairpin structure acts as a nontypical rhoindependent transcriptional terminator (6). The predicted
value of the free energy of its formation was estimated (13) to
be G 20.7 kcal/mol. The calculated value of the parameter d (6) is 24.475, which correlates with a theoretical efficiency of termination of ca. 75%.
To determine the length of the pGA1-coded ctRNA, PctRNA-containing fragments, differing in length, were cloned
into the promoter-probe vector pET2 and promoter activity
was measured in cell extracts of C. glutamicum cells harboring
the individual plasmid constructs. Decrease of activity was obviously an evidence for the presence of a terminator. As shown
in Table 3, a substantial decrease of CAT activity was observed
in C. glutamicum cells harboring plasmid pET2-PctRNA3 carrying the 77-bp P-ctRNA-containing fragment ending just

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Presence () or
absence () of:

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VENKOVA-CANOVA ET AL.

J. BACTERIOL.

downstream of five U residues. This result is in agreement with

the predicted termination features of the above-described hairpin structure (T). Two constructs with longer P-ctRNA-containing fragments (pET2-PctRNA4 and pET2-PctRNA5)
showed still more severe decrease of CAT activity. According
to the results shown in Table 3, the size of ctRNA was estimated to be about 89 nt. The 3 ends of the tested fragments
are shown in Fig. 1.
Since RNA polymerase (RNAP) from C. glutamicum is not
available, we have used RNAP from E. coli (Roche) for in vitro
synthesis of pGA1-coded ctRNA. The reaction was performed
according to del Solar and Espinosa (8, 9). A single major
RNA band was detected on the polyacrylamide gel as a product of the runoff in vitro assay, which proved recognition of the

TABLE 3. Promoter activity of P-ctRNA-containing fragments

differing in length
Plasmid

pET2
pET2-PctRNA1
pET2-PctRNA2
pET2-PctRNA3
pET2-PctRNA4
pET2-PctRNA5

Size of
ctRNA
(nt)

38
64
77
85
89

MIC in C.
glutamicum
(g of Cm/
ml)b

5
90
90
60
30
30

Mean CAT activityc

in C. glutamicum Termination
(mol min1 mg
(%)
of protein1) SD

0.001
0.36 0.002
0.38 0.009
0.078 0.001
0.022 0.003
0.013 0.003

0
0
78
94
96

a
The 3 ends of the cloned fragments in plasmids pET2-PctRNA1, pET2PctRNA2, pET2-PctRNA3, pET2-PctRNA4, and pET2-PctRNA5 are shown in
Fig. 1.
b
Cm, chloramphenicol.
c
Values represent the means from at least four independent experiments.

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FIG. 3. (A) Computer-predicted secondary structure of pGA1-encoded ctRNA. The length of the putative ctRNA is given according to the
results of measuring promoter activity of promoter-containing P-ctRNA fragments, which differ in length. S1, the first hairpin, which might be
formed downstream of the 5end of pGA1-ctRNA; T, the hairpin that resembles rho-independent transcriptional terminators. The sequence of the
U-turn in S1, similar to the U-turn loop consensus motif in some prokaryotic antisense RNAs or in their target RNAs, is given in bold. The
sequence complementary to the putative Shine-Dalgarno sequence of pGA1 rep mRNA is marked by vertical lines. (B) Similarity of the sequence
of pGA1-encoded ctRNA U-turn to the U-turn loop consensus motif in some prokaryotic antisense RNAs or their target RNAs ( ) (according
to Franch et al. [12]). Y, pyrimidine; U, uracil; N, any base, with preference for purine (most often G); R, purine; the nucleotides forming the
top-stem base pairs closing the loops are underlined. chr., chromosome. (C) Computer-predicted secondary structure of the RNA encoded by the
rep gene leader region of plasmid pGA1. SD, putative Shine-Dalgarno site (underlined) of rep gene. The start codon of rep gene is given in boldface
letters.

PLASMID pGA1 FROM CORYNEBACTERIUM GLUTAMICUM

VOL. 185, 2003

TABLE 4. Promoter activity of Prep-containing fragments differing

in length

Plasmid

a
b

1
2
2

MIC in C.
glutamicum
(g of Cm/
ml)a

Mean CAT activityb

in C. glutamicum
(mol min1 mg
of protein1) SD

5
60
20
20
20

0.001
0.147 0.007
0.012 0.002
0.014 0.003
0.011 0.001

Cm, chloramphenicol.
Values represent the means from at least four independent experiments.

promoter P-ctRNA by the E. coli RNAP. However, position of

this band on the gel (RNAs of defined size were used as size
markers) corresponded to the smaller size of ctRNA (65 nt)
(data not shown) in comparison with that estimated in vivo in
C. glutamicum cells (89 nt). The smaller ctRNA, determined by
in vitro assay, could be formed due to different recognition of
ctRNA transcriptional terminator (T) by E. coli RNAP since
its structure differs from that of the typical E. coli rho-independent transcriptional terminators.
Influence of the sequence of the rep leader region on promoter Prep activity. Activity of the promoter of the pGA1 rep
gene, Prep, was found to be very low when the fragment containing also the promoter P-ctRNA was used (plasmid pET2Prep4 in Table 4). To test whether this low activity of Prep was
caused by synthesis of ctRNA, a DNA fragment with a deletion
of the 10 and 35 hexamers of P-ctRNA, which abolishes
synthesis of ctRNA, was cloned into the promoter-probe vector pET2 (plasmid pET2-Prep3). As shown in Table 4, this
deletion had no positive effect on transcription from Prep. A
similarly low CAT activity (0.009 0.001 mol min1. mg of
protein1) was measured in C. glutamicum harboring the plasmid pET2-Prep4(PctRNAmut) in which the mutation abolishing function of the 10 hexamer of P-ctRNA was introduced.
However, when almost the whole rep leader region (90 of 93
nt) was deleted (plasmid pET2-Prep1), a 10-fold increase of
Prep activity was observed (Table 4). Thus, the sequence of the
rep leader region itself seems to act as another regulatory
element negatively influencing expression of the pGA1 rep
gene, probably due to the formation of two distinct secondary
structures (hairpins) on the pGA1 rep mRNA (Fig. 3C). Results with plasmid pET2-Prep2 (Table 4), in which the sequence causing the second hairpin was deleted in the cloned
pGA1 fragment, proved that the presence of the rep leader
sequence responsible for the first (longer) hairpin (designated
IR1 in Fig. 1, estimated value of G 18.5 kcal/mol) is
sufficient to ensure low activity of the Prep. Very similar results
(high activity of Prep in pET2-Prep1 and low activity of Prep in
pET2-Prep2 and in pET2-Prep3) were obtained when promoter
activity was measured in C. glutamicum harboring also plasmid
pSG48 or in E. coli (data not shown). No interaction of the
hairpin(s) with any effector encoded by plasmid pGA1 or by C.
glutamicum chromosome is thus obvious.

DISCUSSION
Plasmid pGA1 from C. glutamicum, classified in the fifth
group of RCR plasmids (26), was found to code for a unique
regulatory element, the Per protein, that positively influences
plasmid copy number and maintenance (22). Although the per
gene expression is negatively controlled at the transcriptional
level by pGA1 gene product(s) (22), this single regulatory
mechanism does not seem to be sufficient to ensure the optimum plasmid copy number in C. glutamicum cells. Indeed, we
have found two other negatively acting regulatory elements,
both influencing expression of the rep gene coding initiator of
pGA1 replication by rolling circle mode. As described here,
the function of these two elements is necessary for negative
control of plasmid pGA1 replication.
The negative effect of the pGA1 rep leader region, present
on a compatible plasmid, on the maintenance of a pGA1 derivative was observed. It indicates that a trans-acting regulatory
element is encoded by this region. We suggest that this transacting regulatory element is a ctRNA since (i) a strong constitutive promoter (designated P-ctRNA) and (ii) a GC-rich sequence, 36 bp downstream of the 1 base of the transcript,
with potential to generate a secondary structure that could
serve as a rho-independent transcriptional terminator, were
found in this region in orientation opposite to the rep gene (see
Fig. 1 and 2). This notion was strongly supported by the presence of a U-turn loop in the first potential hairpin structure
(S1) of the putative ctRNA with a sequence highly similar to
the consensus sequence of U-turns in prokaryotic antisense
RNAs or in their complementary target RNAs (Fig. 3A and
B). This U-turn loop is considered to promote pairing with the
complementary sequence on the rep mRNA (Fig. 3C). Efficient
interaction of pGA1-encoded ctRNA with the rep mRNA can
be predicted also from its other structural features typical for
regulatory ctRNAs (15, 16), namely, from the size of the S1
loop (7 nt) and from presence of an internal loop in the S1
stem (see Fig. 3A). According to the approximate size of the
transcript starting from P-ctRNA (ca. 89 nt), determined by
measuring promoter activity of fragments differing in length,
the end of pGA1-encoded ctRNA seems to correlate with start
of the rep mRNA (see Table 3 and Fig. 1). The smaller ctRNA
(65 nt) was detected by in vitro assay using E. coli RNAP. It
was found that terminators of C. glutamicum often differ from
those of E. coli both by structure and function (3). Therefore,
the smaller size of the in vitro ctRNA transcript was caused
very probably by transcriptional termination driven by the heterologous RNAP differing in its action from that of the native
RNAP.
Strong positive influence of inactivation of promoter
P-ctRNA on copy number of the low-copy-number pGA1 derivative pKG32 (Table 2) proved that the product of the gene
transcribed from this promoter (i.e., ctRNA) controls negatively number of pGA1 plasmid copies. Inhibition of synthesis
of the Rep protein, a positively acting initiator of pGA1 replication, by interaction of ctRNA with the complementary
leader sequence of the rep mRNA is apparently responsible for
the negative effect of ctRNA on pGA1 copy number. A dramatic increase in pKG32 copy number, observed when inhibitor of the Rep protein synthesis was absent, was thus caused
most probably by the increase of the Rep concentration in C.

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pET2
pET2-Prep1
pET2-Prep2
pET2-Prep3
pET2-Prep4

No. of
Presence ()
secondary
or absence
structures
() of
on rep
ctRNA
mRNA

2407

2408

VENKOVA-CANOVA ET AL.

serves as a basis for further studies on mechanisms of Per

function.
ACKNOWLEDGMENTS
We are grateful to Manuel Espinosa and Gloria del Solar for helpful
advice and for critical reading of the manuscript.
This work was supported by grant 204/01/0998 from the Grant
Agency of the Czech Republic and by Institutional Research Concept
grant no. AV0Z5020903.
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glutamicum cells. These results suggest the rate-limiting role of

Rep for pGA1 replication, which is in agreement with data
described in other RCR plasmids (4). A simultaneously observed positive effect of inactivation of promoter P-ctRNA on
segregational stability of unstable plasmid pKG32 reflects most
probably correlation between plasmid copy number and maintenance, which might be common for small plasmids lacking a
particular stabilizing system.
Unlike the observed positive effect of inactivation of promoter P-ctRNA on pGA1 copy number, inactivation of this
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(Table 4). These results indicate that the pGA1-encoded
ctRNA acts negatively at the posttranscriptional level. The
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plasmid pGA1 copies. It was found that the presence of the
sequence IR1 (Fig. 1) in the rep leader region is responsible for
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ctRNA complementary to the rep mRNA leader region. Thus,
the IR1 sequence is a second regulatory element, in addition to
the ctRNA, negatively influencing expression of the pGA1 rep
gene. Observation that no trans-acting element encoded by the
plasmid pGA1 or by C. glutamicum chromosome influenced
the negative effect of the IR1 sequence on the Prep activity
excluded functioning of this sequence as a DNA target (operator) for any effector. Therefore, it seems that it is the distinct
secondary structure of IR1 formed within the leader region of
rep mRNA, which is responsible for the negative effect on the
rep gene expression. Interference of this structure with the
function of RNA polymerase may be one of the possible mechanisms. A regulatory role of such structure(s) in control of
number of plasmid copies has not yet been described in any
bacterial plasmid.
As proved previously, maintaining of the optimum number
of pGA1 copies in C. glutamicum cells also requires the functioning of the positively acting element, the Per protein (22).
When possible mechanisms of its activity were tested, it was
found that, regardless its positive effect on pGA1 copy number,
it has no positive effect on the activity of promoter Prep, and no
binding of this protein to pGA1 DNA was observed (T. Venkova-Canova, unpublished results). As shown in this study,
abolishment of ctRNA synthesis also has a strong positive
effect on plasmid copy number but no effect on activity of
promoter Prep. Taking these two findings together, we may
suggest that both copy number controlling elements (i.e., the
positively acting Per protein and the negatively acting ctRNA)
function at the posttranscriptional level. The positive effect of
Per on the pGA1 copy number might be explained by its
interaction with ctRNA, resulting in a decrease of negative
effect of ctRNA on the rep gene expression. This hypothesis

J. BACTERIOL.

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