- Email: [email protected]
Cloning and characterization of the recA gene of Paracoccus denitrificans and construction of a recA-deficient mutant1
FEMS Microbiology Letters 147 (1997) 209^213
Cloning and characterization of the recA gene of Paracoccus denitri¢cans and construction of a recA-de¢cient mutant Antonio R. Fernandez de Henestrosa, Alfonso del Rey, Rauël Tarragoè, Jordi Barbeè * Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain
Received 5 November 1996; revised 10 December 1996; accepted 11 December 1996
Abstract
The
gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy. recA
sphaeroides recA
Keywords : recA
gene;
Paracoccus denitri¢cans
;
recA
mutant ;
Rhizobium etli
1. Introduction
Paracoccus denitri¢cans is a Gram-negative aerobic soil bacterium able to grow under a variety of conditions. Taxonomically, Paracoccus has been grouped within the K-subdivision of the eubacterial kingdom where, together with Rhodobacter capsulatus and Rhodobacter sphaeroides, it forms a separate group [1]. The physiological versatility of P. denitri¢cans has stimulated the interest in the development of the genetics and molecular biology of this organism to further progress in both basic bioenergetic
* Corresponding author. Tel.: +34 (3) 581 1837; fax: +34 (3) 581 2387; e-mail: [email protected] This article is dedicated to Dr. Berta Clerch, who died suddenly on 18 September 1996.
;
Rhodobacter sphaeroides
and biotechnological ¢elds. In this sense, many genes of this organism have been isolated and characterized in recent years [2]. Likewise, several mutants and vectors to enable the genetic manipulation of P. denitri¢cans have been constructed [2]. Frequently, genetic studies of any bacterial species require the use of recombination (Rec3 ) mutants. Such variants have been useful, among other applications, in complementation analysis, in studies with cloned genes, ensuring the stability of expression vectors, and in vivo construction of R-prime plasmids. In Escherichia coli the key element in homologous recombination, as well as in the SOS response, DNA repair and mutagenesis, is the recA gene [3]. More than 60 recA analogous genes have been cloned from di¡erent bacteria by several methods [4], among them those of R. capsulatus and R. sphaeroides
0378-1097 / 97 / $17.00 Copyright ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 5 2 6 - 5
FEMSLE 7415 14-5-97
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213
210
P. denitri¢cans in BHI [7], R. etli R. sphaeroides and R. capsulatus
[5,6]. At this point, it is worth noting that although
grown in LB [7],
these two phototrophic bacteria are very close from
in PY broth [7], and
a phylogenetic and metabolic point of view, the reg-
in 290A medium [6]. Antibiotics were added to the
ulation of their
recA
genes is not reciprocal [6].
culture media at the appropriate concentration for
In this context, the aim of this work has been
each bacterial species [8].
double. First of all, the isolation and sequencing of
recA
the
3
gene
P. denitri¢cans
of
to
construct a
derivative to improve its genetic manipula-
RecA
2.2. Genetic, biochemical methods and DNA techniques
tion system. Secondly, the analysis of the expression of the
P. denitri¢cans recA
gene in comparison to
Triparental matings using pRK2013 as the mobi-
of the Proteo-
lizing plasmid were as previously reported [6]. UV
bacteria to further understand what the SOS system
irradiation and mitomycin C treatment were per-
K-subdivision
other members of the
is like in these organisms.
formed as cited [5].
P. denitri¢cans
chromosome ex-
change markers were done as described [9]. All DNA methodology was as published [10]. The entire nucleotide sequence was determined with both DNA
2. Materials and methods
strands by the dideoxy method on an ALF Se-
2.1. Bacterial strains and cultivation conditions
quencer
(Pharmacia
Biotech).
Computer
analysis
was carried out with the University of Wisconsin The bacterial strains and plasmids used in this
E. coli
work are listed in Table 1.
was normally
Genetics Computer Group package (version 8.0). The nucleotide sequence of the
P. denitri¢cans recA
Table 1 Bacterial strains and plasmids Organism
Relevant features
Source or
or plasmid E. coli
MC1061
supE4, vlacU169 vlacX74 hsdR
S17-1 (
As S17-1 lysogenized with
DH5
K
reference
Vpir)
(Ò80
lacZ
vM15),
Vpir
hasdR17, recA1, endA1, gyrA 96, thi-1, relA1
[10] M. Casadaban
bacteriophage
[12]
P. denitri¢cans r
Pd1222
Rif , enhanced conjugation frequence
[13]
UAP10
As Pd1222 but
This study
recA : : 6Km
Plasmids r
r
r
pSUP202
Cm
pRK2013
Tra
pHP45
Source of Km
6Km
Tc
Ap
Mob
of RK2 ColE1 replicon, Km r
M. Vicente r
D.R. Helinski
cassette
M. Krisch
lacZ
A promoterless vector useful to make
pBluescript SK(+)
Ap
pGEM-T
Ap
pLV106
Tc
[14]
pUA600
R. etli recA-lacZ fusion pBSK(+) carrying P. denitri¢cans recA gene in a 3-kb EcoRI fragment As pSUP202 containing recA : : 6Km of P. denitri¢cans As pUJ8 carrying a 250-bp EcoRI-BamHI fragment containing the recA P. denitri¢cans pLV106 carrying P. denitri¢cans recA-lacZ fusion pLV106 carrying R. capsulatus recA-lacZ fusion pLV106 carrying R. spharoides recA-lacZ fusion
[11]
pUA617 pUA618 pUA620
pUA621 pUA622 pUA623
fusions, Ap
r
pUJ8
[12]
r
Stratagene
r
Promega
r
pLV106 carrying
FEMSLE 7415 14-5-97
This study This study promoter from
This study
This study This study This study
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213
Fig. 1. UV sensitivity of RecA (E) and RecA3 (F) strains of P. denitri¢cans Pd1222. The fraction of surviving cells after irradiation with increasing amounts of UV radiation was determined by comparison with non-irradiated cells.
gene and its £anking regions will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under accession number U59631. 3. Results and discussion
3.1. Cloning of the P. denitri¢cans recA gene
It is has been established that RecA proteins from di¡erent bacteria are highly similar in their amino acid sequences [4]. Since P. denitri¢cans and R. sphaeroides are closely related from a phylogenetic point of view [1], and their genomes present the same range of G+C content (64^67%), we predicted that the recA genes from these bacteria should possess a high degree of identity in their nucleotide sequences. To con¢rm this hypothesis, several highstringency Southern blots were performed with chromosomal DNA of P. denitri¢cans Pd1222 individually digested with EcoRI, PstI or SmaI. The probe used was a digoxigenin random-labeled 1.7-kb SmaI chromosomal fragment containing the R. sphaeroides recA gene. For each digestion, one band hybridized with the probe (data not shown). The band size was s 6 kb for EcoRI, s 23 kb for PstI and s 9 kb for SmaI. This result suggests that a recA-like gene related to that of R. sphaeroides is contained in P. denitri¢cans.
To isolate the P. denitri¢cans recA gene, a gene bank of this organism, in the VGEM12 vector, was
211
probed with the 1.7-kb SmaI fragment harboring the R. sphaeroides recA gene. Among 2000 plaques screened, 15 reacted with the probe. One of these positive phages was further analyzed by restriction enzyme digestion and Southern blot hybridization. A 3-kb EcoRI fragment of this phage, which hybridizes with the R. sphaeroides probe, was isolated and ligated into the unique EcoRI site of the pBluescript SK(+) plasmid. The clone obtained, pUA617, was tested again with the R. sphaeroides recA gene probe to con¢rm that the P. denitri¢cans recA gene was contained in it. 3.2. Sequence analysis of the P. denitri¢cans recA gene
The nucleotide sequence of the P. denitri¢cans 3kb EcoRI revealed one large open reading frame, which was four nucleotides from a typical Shine-Dalgarno sequence (GAGGA). The P. denitri¢cans recA ORF of 1071 nucleotides codes for a polypeptide of 356 amino acids whose calculated molecular mass is 38 097 Da. The P. denitri¢cans recA is 3 nucleotides longer than that of R. sphaeroides (not shown). Comparison of the P. denitri¢cans RecA with the RecA proteins of R. sphaeroides, R. capsulatus, R. etli and E. coli showed identity levels of 88.6%, 85.6%, 72.9% and 64.3%, respectively, in the deduced amino acid sequence. The greatest degree of di¡erence between these proteins is found primarily in the C-termini. Amino acid residues in E. coli RecA associated with functional activities which include ATP fold, ATP binding, protease activity, and homologous recombination, as well as the RecA signature peptide [4], are all conserved in the RecA of P. denitri¢cans. However, upstream of the coding region of the P. denitri¢cans recA gene there was no sequence homologous to an E. coli LexA binding site (SOS box), suggesting that the regulation of this P. denitri¢cans gene may di¡er from that of the E. coli gene. 3.3. Construction and characterization of a recA mutant
To obtain a recA mutant of P. denitri¢cans, a 2-kb kanamycin-resistant cassette was inserted into the internal BstXI site of the P. denitri¢cans recA gene,
FEMSLE 7415 14-5-97
212
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213
Fig. 2. DNA damage-mediated induction of the recA-lacZ fusion from P. denitri¢cans (pUA621 plasmid) contained in RecA (E) and RecA3 (F) strains of this organism. The induction of the recA-lacZ fusions of R. capsulatus (a), R. sphaeroides (b) and R. etli (R) contained in pUA622, pUA623 and pUA600 plasmids, respectively, is also shown. In all cases, the DNA-damaging agent used was mitomycin C at 10 Wg ml31 . After mitomycin C addition, periodic samples were taken from exponentially growing cultures and assayed for L-galactosidase activity as reported [7]. The induction factor is, for each case, the ratio between speci¢c units of L-galactosidase of the treated cells and the same parameter of the untreated cells. All values are reproducible to within an error of þ 10%.
which had previously been cloned in the pSUP202 plasmid, giving rise to pUA618. This plasmid was transferred to a Rifr derivative of P. denitri¢cans Pd1222 as reported [6]. Kanamycin-resistant transconjugants were screened for loss of vector-mediated tetracycline resistance to detect putative mutants which had exchanged their wild-type gene for the inactivated recA as a consequence of a double crossover event. For one such strain (UAP10), this was unequivocally con¢rmed by Southern experiments by using the 3-kb EcoRI chromosomal fragment containing the P. denitri¢cans recA as a probe (data not shown). The recA mutant was extremely sensitive to UV irradiation (Fig. 1). With increasing dosages of UV, the survival rate decreased drastically. Thus, at a dosage higher than 40 J m32 , survival was lower than 1035 % (Fig. 1). On the other hand, wild-type cells UV-irradiated at the same doses presented a survival 104 -fold higher. 3.4. Expression of the P. denitri¢cans recA gene
To analyze the expression of the P. denitri¢cans , a fragment containing 228 bp of its promoter
recA
region, as well as 23 bp of its encoding region, was obtained from plasmid pUA617 by PCR ampli¢cation using 5P-GAATTCAGCCATTGCGGCACGC3P and 5P-GGATCCTCGAAAAGTGTTGCCCC-3P as upper and lower primers. These primers are centered at positions 3224 and +17 with respect to the ¢rst nucleotide of the predicted translational starting codon of the P. denitri¢cans recA gene. Afterwards, the 251-bp fragment obtained was cloned into the EcoRI-BamHI digested pUJ8 plasmid, originating a fusion between the recA region and the promoterless trpP-lacZ gene. The plasmid carrying this construction was designated pUA620. The NotI-end¢lled fragment of pUA620 containing the recAlacZ fusion was then inserted into the unique SmaI cloning site of the broad-range host pLV106 plasmid, giving rise to pUA621, which was introduced into both RecA and RecA3 strains of P. denitri¢cans. Fig. 2 shows that expression of the P. denitri¢cans recA gene is enhanced in wild-type mitomycin Ctreated cells but not in the RecA3 mutant. This indicates that a regulatory element is present upstream of the coding region of the P. denitri¢cans recA gene and suggests that P. denitri¢cans must possess a LexA-like repressor capable of blocking recA expression in undamaged cells and of being inactivated by RecA co-protease. As has been cited above, P. denitri¢cans, R. sphaeroides, R. capsulatus and R. etli belong to the K-subdivision of the Proteobacteria group. Thus, and to determine the relationship existing between the SOS systems of these organisms, three plasmids containing the promoter region of the recA gene of R. sphaeroides, R. capsulatus and R. etli fused to the lacZ gene were introduced in P. denitri¢cans and assayed by L-galactosidase production in the presence of mitomycin C. Surprisingly, only the R. etli recA gene was induced in these conditions (Fig. 2). These results, together with those previously reported, indicate that the recA genes of P. denitri¢cans, R. sphaeroides and R. capsulatus have di¡erent regulatory sequences despite the high identity existing between their encoded products. Moreover, the fact that the R. etli recA gene was DNA damageinducible in the P. denitri¢cans genetic background suggests that the recA of these two organisms may be under either the same or a very close regulatory el-
FEMSLE 7415 14-5-97
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213 ement. In this respect, it is worth noting that up-
recA
stream of the
etli
genes of
P. denitri¢cans
R.
and
there are two very similar sequences displaying
dyad
respectively. DNA
recA
TTGN10 CAA
symmetry, In
fact,
it
has
damage-mediated
TTGN11 CAA,
and
been
shown
expression
of
that
the
the
R. etli
gene is dependent upon this TTGN11 CAA pal-
indrome [11]. Finally, the introduction of a
recA
213
[3] Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in
Escherichia coli.
Microbiol.
Rev. 48, 60^93. [4] Karlin, S. and Brocchieri, L. (1996) Evolutionary conservation of
recA
genes in relation to protein structure and function.
J. Bacteriol. 178, 1881^1894. è ndez [5] Ferna (1995) and
de
Henestrosa,
Non-reciprocal
A.R.,
regulation
Rivera, of
Rhodobacter sphaeroides recA
E.
and
è, Barbe
J.
Rhodobacter capsulatus
genes expression. FEMS
Microbiol. Lett. 129, 175^182.
knockout mu-
[6] Calero,
S.,
Fernandez
de
Henestrosa,
A.R.
and
è, Barbe
J.
tation into the Pd1222 strain, which presents an en-
(1994) Molecular cloning, sequence and regulation of expres-
hanced conjugation frequency, will help in the devel-
sion of the
opment
of
a
stable
host-vector
organism. Furthermore, this
recA
system
in
this
mutant will allow
detailed analysis of the molecular basis of recombi-
P. denitri¢cans.
nation in
recA gene of the phototrophic bacterium Rhodobacter sphaeroides. Mol. Gen. Genet. 242, 116^120.
[7] Miller, J.H. (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [8] Miller, J.H. (1991) Bacterial genetic systems. Methods Enzymol. 204. [9] vanHaute, E., Joos, H., Maes, M., Warren, G., vanMontagu, M. and Schell, J. (1983) Intergeneric transfer and exchange
Acknowledgments
recombination of restriction fragments cloned in pBR322 : a novel strategy for the reversal genetics of the Ti plasmids of
Agrobacterium tumefaciens. This work was funded by Grant PB94-0687 of the èn Direccio è cnica Te
General
of
Spain
de
èn Investigacio
(DGICYT),
and
è¢ca Cient| partially
y
sup-
ported by the Comissionat per Universitats i Recerca de la Generalitat de Catalunya. We gratefully ac-
EMBO J. 2, 411^417.
[10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [11] Tapias, (1996)
A.,
è ndez Ferna
Characterization
etli recA
de of
Henestrosa, the
A.R.
promoter
of
and the
è, Barbe
J.
Rhizobium
gene. J. Bacteriol. (in press).
è General d'Uniknowledge the help of the Direccio
[12] De Lorenzo, V., Herrero, M., Jakubzik, U. and Timmis, K.N.
versitats de la Generalitat de Catalunya for di¡erent
(1990) Mini-Tn5 transposon derivatives for insertion mutagenesis,
grants for the purchase of equipment.
promoter
probing
and
chromosomal
insertion
of
cloned DNA in Gram-negative eubacteria. J. Bacteriol. 172, 6568^6572. [13] De Vries, G.E., Harms, N., Hoogendijk, J. and Stouthamer,
References
A.H. (1989) Isolation and characterization of
nitri¢cans
[1] Woese, C.R., Stackebrandt, E., Weisburg, W.G., Paster, B.J., Madigan, M.T., Fowler, V.J., Hahn, C.M., Blanz, P., Gupta, R., Nealson, K.H. and Fox, G.E. (1984) The phylogeny of purple bacteria : the alpha subdivision. Syst. Appl. Microbiol.
[2] Steinru ë cke, P. and Ludwig, B. (1993) Genetics of
denitri¢cans.
pleiotropic
loss
of
a
n(GATC)n
DNA-modifying
property.
Arch. Microbiol. 152, 52^57. [14] Lee, J.K. and Kaplan, S. (1992)
cis-Acting regulatory elements puc operon transcrip-
involved in oxygen and light control of tion in
5, 315^326.
Paracoccus de-
mutants with increased conjugation frequencies and
Paracoccus
FEMS Microbiol. Rev. 104, 83^118.
FEMSLE 7415 14-5-97
Rhodobacter sphaeroides.
J. Bacteriol. 174, 1146^1157.
Cloning and characterization of the recA gene of Paracoccus denitri¢cans and construction of a recA-de¢cient mutant Antonio R. Fernandez de Henestrosa, Alfonso del Rey, Rauël Tarragoè, Jordi Barbeè * Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain
Received 5 November 1996; revised 10 December 1996; accepted 11 December 1996
Abstract
The
gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy. recA
sphaeroides recA
Keywords : recA
gene;
Paracoccus denitri¢cans
;
recA
mutant ;
Rhizobium etli
1. Introduction
Paracoccus denitri¢cans is a Gram-negative aerobic soil bacterium able to grow under a variety of conditions. Taxonomically, Paracoccus has been grouped within the K-subdivision of the eubacterial kingdom where, together with Rhodobacter capsulatus and Rhodobacter sphaeroides, it forms a separate group [1]. The physiological versatility of P. denitri¢cans has stimulated the interest in the development of the genetics and molecular biology of this organism to further progress in both basic bioenergetic
* Corresponding author. Tel.: +34 (3) 581 1837; fax: +34 (3) 581 2387; e-mail: [email protected] This article is dedicated to Dr. Berta Clerch, who died suddenly on 18 September 1996.
;
Rhodobacter sphaeroides
and biotechnological ¢elds. In this sense, many genes of this organism have been isolated and characterized in recent years [2]. Likewise, several mutants and vectors to enable the genetic manipulation of P. denitri¢cans have been constructed [2]. Frequently, genetic studies of any bacterial species require the use of recombination (Rec3 ) mutants. Such variants have been useful, among other applications, in complementation analysis, in studies with cloned genes, ensuring the stability of expression vectors, and in vivo construction of R-prime plasmids. In Escherichia coli the key element in homologous recombination, as well as in the SOS response, DNA repair and mutagenesis, is the recA gene [3]. More than 60 recA analogous genes have been cloned from di¡erent bacteria by several methods [4], among them those of R. capsulatus and R. sphaeroides
0378-1097 / 97 / $17.00 Copyright ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 5 2 6 - 5
FEMSLE 7415 14-5-97
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213
210
P. denitri¢cans in BHI [7], R. etli R. sphaeroides and R. capsulatus
[5,6]. At this point, it is worth noting that although
grown in LB [7],
these two phototrophic bacteria are very close from
in PY broth [7], and
a phylogenetic and metabolic point of view, the reg-
in 290A medium [6]. Antibiotics were added to the
ulation of their
recA
genes is not reciprocal [6].
culture media at the appropriate concentration for
In this context, the aim of this work has been
each bacterial species [8].
double. First of all, the isolation and sequencing of
recA
the
3
gene
P. denitri¢cans
of
to
construct a
derivative to improve its genetic manipula-
RecA
2.2. Genetic, biochemical methods and DNA techniques
tion system. Secondly, the analysis of the expression of the
P. denitri¢cans recA
gene in comparison to
Triparental matings using pRK2013 as the mobi-
of the Proteo-
lizing plasmid were as previously reported [6]. UV
bacteria to further understand what the SOS system
irradiation and mitomycin C treatment were per-
K-subdivision
other members of the
is like in these organisms.
formed as cited [5].
P. denitri¢cans
chromosome ex-
change markers were done as described [9]. All DNA methodology was as published [10]. The entire nucleotide sequence was determined with both DNA
2. Materials and methods
strands by the dideoxy method on an ALF Se-
2.1. Bacterial strains and cultivation conditions
quencer
(Pharmacia
Biotech).
Computer
analysis
was carried out with the University of Wisconsin The bacterial strains and plasmids used in this
E. coli
work are listed in Table 1.
was normally
Genetics Computer Group package (version 8.0). The nucleotide sequence of the
P. denitri¢cans recA
Table 1 Bacterial strains and plasmids Organism
Relevant features
Source or
or plasmid E. coli
MC1061
supE4, vlacU169 vlacX74 hsdR
S17-1 (
As S17-1 lysogenized with
DH5
K
reference
Vpir)
(Ò80
lacZ
vM15),
Vpir
hasdR17, recA1, endA1, gyrA 96, thi-1, relA1
[10] M. Casadaban
bacteriophage
[12]
P. denitri¢cans r
Pd1222
Rif , enhanced conjugation frequence
[13]
UAP10
As Pd1222 but
This study
recA : : 6Km
Plasmids r
r
r
pSUP202
Cm
pRK2013
Tra
pHP45
Source of Km
6Km
Tc
Ap
Mob
of RK2 ColE1 replicon, Km r
M. Vicente r
D.R. Helinski
cassette
M. Krisch
lacZ
A promoterless vector useful to make
pBluescript SK(+)
Ap
pGEM-T
Ap
pLV106
Tc
[14]
pUA600
R. etli recA-lacZ fusion pBSK(+) carrying P. denitri¢cans recA gene in a 3-kb EcoRI fragment As pSUP202 containing recA : : 6Km of P. denitri¢cans As pUJ8 carrying a 250-bp EcoRI-BamHI fragment containing the recA P. denitri¢cans pLV106 carrying P. denitri¢cans recA-lacZ fusion pLV106 carrying R. capsulatus recA-lacZ fusion pLV106 carrying R. spharoides recA-lacZ fusion
[11]
pUA617 pUA618 pUA620
pUA621 pUA622 pUA623
fusions, Ap
r
pUJ8
[12]
r
Stratagene
r
Promega
r
pLV106 carrying
FEMSLE 7415 14-5-97
This study This study promoter from
This study
This study This study This study
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213
Fig. 1. UV sensitivity of RecA (E) and RecA3 (F) strains of P. denitri¢cans Pd1222. The fraction of surviving cells after irradiation with increasing amounts of UV radiation was determined by comparison with non-irradiated cells.
gene and its £anking regions will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under accession number U59631. 3. Results and discussion
3.1. Cloning of the P. denitri¢cans recA gene
It is has been established that RecA proteins from di¡erent bacteria are highly similar in their amino acid sequences [4]. Since P. denitri¢cans and R. sphaeroides are closely related from a phylogenetic point of view [1], and their genomes present the same range of G+C content (64^67%), we predicted that the recA genes from these bacteria should possess a high degree of identity in their nucleotide sequences. To con¢rm this hypothesis, several highstringency Southern blots were performed with chromosomal DNA of P. denitri¢cans Pd1222 individually digested with EcoRI, PstI or SmaI. The probe used was a digoxigenin random-labeled 1.7-kb SmaI chromosomal fragment containing the R. sphaeroides recA gene. For each digestion, one band hybridized with the probe (data not shown). The band size was s 6 kb for EcoRI, s 23 kb for PstI and s 9 kb for SmaI. This result suggests that a recA-like gene related to that of R. sphaeroides is contained in P. denitri¢cans.
To isolate the P. denitri¢cans recA gene, a gene bank of this organism, in the VGEM12 vector, was
211
probed with the 1.7-kb SmaI fragment harboring the R. sphaeroides recA gene. Among 2000 plaques screened, 15 reacted with the probe. One of these positive phages was further analyzed by restriction enzyme digestion and Southern blot hybridization. A 3-kb EcoRI fragment of this phage, which hybridizes with the R. sphaeroides probe, was isolated and ligated into the unique EcoRI site of the pBluescript SK(+) plasmid. The clone obtained, pUA617, was tested again with the R. sphaeroides recA gene probe to con¢rm that the P. denitri¢cans recA gene was contained in it. 3.2. Sequence analysis of the P. denitri¢cans recA gene
The nucleotide sequence of the P. denitri¢cans 3kb EcoRI revealed one large open reading frame, which was four nucleotides from a typical Shine-Dalgarno sequence (GAGGA). The P. denitri¢cans recA ORF of 1071 nucleotides codes for a polypeptide of 356 amino acids whose calculated molecular mass is 38 097 Da. The P. denitri¢cans recA is 3 nucleotides longer than that of R. sphaeroides (not shown). Comparison of the P. denitri¢cans RecA with the RecA proteins of R. sphaeroides, R. capsulatus, R. etli and E. coli showed identity levels of 88.6%, 85.6%, 72.9% and 64.3%, respectively, in the deduced amino acid sequence. The greatest degree of di¡erence between these proteins is found primarily in the C-termini. Amino acid residues in E. coli RecA associated with functional activities which include ATP fold, ATP binding, protease activity, and homologous recombination, as well as the RecA signature peptide [4], are all conserved in the RecA of P. denitri¢cans. However, upstream of the coding region of the P. denitri¢cans recA gene there was no sequence homologous to an E. coli LexA binding site (SOS box), suggesting that the regulation of this P. denitri¢cans gene may di¡er from that of the E. coli gene. 3.3. Construction and characterization of a recA mutant
To obtain a recA mutant of P. denitri¢cans, a 2-kb kanamycin-resistant cassette was inserted into the internal BstXI site of the P. denitri¢cans recA gene,
FEMSLE 7415 14-5-97
212
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213
Fig. 2. DNA damage-mediated induction of the recA-lacZ fusion from P. denitri¢cans (pUA621 plasmid) contained in RecA (E) and RecA3 (F) strains of this organism. The induction of the recA-lacZ fusions of R. capsulatus (a), R. sphaeroides (b) and R. etli (R) contained in pUA622, pUA623 and pUA600 plasmids, respectively, is also shown. In all cases, the DNA-damaging agent used was mitomycin C at 10 Wg ml31 . After mitomycin C addition, periodic samples were taken from exponentially growing cultures and assayed for L-galactosidase activity as reported [7]. The induction factor is, for each case, the ratio between speci¢c units of L-galactosidase of the treated cells and the same parameter of the untreated cells. All values are reproducible to within an error of þ 10%.
which had previously been cloned in the pSUP202 plasmid, giving rise to pUA618. This plasmid was transferred to a Rifr derivative of P. denitri¢cans Pd1222 as reported [6]. Kanamycin-resistant transconjugants were screened for loss of vector-mediated tetracycline resistance to detect putative mutants which had exchanged their wild-type gene for the inactivated recA as a consequence of a double crossover event. For one such strain (UAP10), this was unequivocally con¢rmed by Southern experiments by using the 3-kb EcoRI chromosomal fragment containing the P. denitri¢cans recA as a probe (data not shown). The recA mutant was extremely sensitive to UV irradiation (Fig. 1). With increasing dosages of UV, the survival rate decreased drastically. Thus, at a dosage higher than 40 J m32 , survival was lower than 1035 % (Fig. 1). On the other hand, wild-type cells UV-irradiated at the same doses presented a survival 104 -fold higher. 3.4. Expression of the P. denitri¢cans recA gene
To analyze the expression of the P. denitri¢cans , a fragment containing 228 bp of its promoter
recA
region, as well as 23 bp of its encoding region, was obtained from plasmid pUA617 by PCR ampli¢cation using 5P-GAATTCAGCCATTGCGGCACGC3P and 5P-GGATCCTCGAAAAGTGTTGCCCC-3P as upper and lower primers. These primers are centered at positions 3224 and +17 with respect to the ¢rst nucleotide of the predicted translational starting codon of the P. denitri¢cans recA gene. Afterwards, the 251-bp fragment obtained was cloned into the EcoRI-BamHI digested pUJ8 plasmid, originating a fusion between the recA region and the promoterless trpP-lacZ gene. The plasmid carrying this construction was designated pUA620. The NotI-end¢lled fragment of pUA620 containing the recAlacZ fusion was then inserted into the unique SmaI cloning site of the broad-range host pLV106 plasmid, giving rise to pUA621, which was introduced into both RecA and RecA3 strains of P. denitri¢cans. Fig. 2 shows that expression of the P. denitri¢cans recA gene is enhanced in wild-type mitomycin Ctreated cells but not in the RecA3 mutant. This indicates that a regulatory element is present upstream of the coding region of the P. denitri¢cans recA gene and suggests that P. denitri¢cans must possess a LexA-like repressor capable of blocking recA expression in undamaged cells and of being inactivated by RecA co-protease. As has been cited above, P. denitri¢cans, R. sphaeroides, R. capsulatus and R. etli belong to the K-subdivision of the Proteobacteria group. Thus, and to determine the relationship existing between the SOS systems of these organisms, three plasmids containing the promoter region of the recA gene of R. sphaeroides, R. capsulatus and R. etli fused to the lacZ gene were introduced in P. denitri¢cans and assayed by L-galactosidase production in the presence of mitomycin C. Surprisingly, only the R. etli recA gene was induced in these conditions (Fig. 2). These results, together with those previously reported, indicate that the recA genes of P. denitri¢cans, R. sphaeroides and R. capsulatus have di¡erent regulatory sequences despite the high identity existing between their encoded products. Moreover, the fact that the R. etli recA gene was DNA damageinducible in the P. denitri¢cans genetic background suggests that the recA of these two organisms may be under either the same or a very close regulatory el-
FEMSLE 7415 14-5-97
A.R. Fernandez de Henestrosa et al. / FEMS Microbiology Letters 147 (1997) 209^213 ement. In this respect, it is worth noting that up-
recA
stream of the
etli
genes of
P. denitri¢cans
R.
and
there are two very similar sequences displaying
dyad
respectively. DNA
recA
TTGN10 CAA
symmetry, In
fact,
it
has
damage-mediated
TTGN11 CAA,
and
been
shown
expression
of
that
the
the
R. etli
gene is dependent upon this TTGN11 CAA pal-
indrome [11]. Finally, the introduction of a
recA
213
[3] Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in
Escherichia coli.
Microbiol.
Rev. 48, 60^93. [4] Karlin, S. and Brocchieri, L. (1996) Evolutionary conservation of
recA
genes in relation to protein structure and function.
J. Bacteriol. 178, 1881^1894. è ndez [5] Ferna (1995) and
de
Henestrosa,
Non-reciprocal
A.R.,
regulation
Rivera, of
Rhodobacter sphaeroides recA
E.
and
è, Barbe
J.
Rhodobacter capsulatus
genes expression. FEMS
Microbiol. Lett. 129, 175^182.
knockout mu-
[6] Calero,
S.,
Fernandez
de
Henestrosa,
A.R.
and
è, Barbe
J.
tation into the Pd1222 strain, which presents an en-
(1994) Molecular cloning, sequence and regulation of expres-
hanced conjugation frequency, will help in the devel-
sion of the
opment
of
a
stable
host-vector
organism. Furthermore, this
recA
system
in
this
mutant will allow
detailed analysis of the molecular basis of recombi-
P. denitri¢cans.
nation in
recA gene of the phototrophic bacterium Rhodobacter sphaeroides. Mol. Gen. Genet. 242, 116^120.
[7] Miller, J.H. (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [8] Miller, J.H. (1991) Bacterial genetic systems. Methods Enzymol. 204. [9] vanHaute, E., Joos, H., Maes, M., Warren, G., vanMontagu, M. and Schell, J. (1983) Intergeneric transfer and exchange
Acknowledgments
recombination of restriction fragments cloned in pBR322 : a novel strategy for the reversal genetics of the Ti plasmids of
Agrobacterium tumefaciens. This work was funded by Grant PB94-0687 of the èn Direccio è cnica Te
General
of
Spain
de
èn Investigacio
(DGICYT),
and
è¢ca Cient| partially
y
sup-
ported by the Comissionat per Universitats i Recerca de la Generalitat de Catalunya. We gratefully ac-
EMBO J. 2, 411^417.
[10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [11] Tapias, (1996)
A.,
è ndez Ferna
Characterization
etli recA
de of
Henestrosa, the
A.R.
promoter
of
and the
è, Barbe
J.
Rhizobium
gene. J. Bacteriol. (in press).
è General d'Uniknowledge the help of the Direccio
[12] De Lorenzo, V., Herrero, M., Jakubzik, U. and Timmis, K.N.
versitats de la Generalitat de Catalunya for di¡erent
(1990) Mini-Tn5 transposon derivatives for insertion mutagenesis,
grants for the purchase of equipment.
promoter
probing
and
chromosomal
insertion
of
cloned DNA in Gram-negative eubacteria. J. Bacteriol. 172, 6568^6572. [13] De Vries, G.E., Harms, N., Hoogendijk, J. and Stouthamer,
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