Gene. 121 (1992) 133-136 0 1992 Elsevier Science
GENE
Publishers
B.V. All rights reserved.
133
0378-l 119/92/$05.00
06720
analysis of the recA gene of Agrobacterium tumefaciens C58
Molecular (Homologous
recombination;
H. Wardhan”, Depurtrnenr.~
M.J. McPhersonb,
nucleotide-binding
Carol A. Harris’,
domain)
Ela Sharmad
and G.R.K.
Sastry”
14‘IGenerics und ” Biochemistr_v and h Biotechnology Unit, University of Leeds. Leeds. UK: and ’ Department of Biochemistry. GIrtso Group Re-
search Ltd., Greenfbrd. Middlesex. Received
SOS regulation;
by T.D. McKnight:
UK
22 August
1991; Accepted:
22 September
1991; Received
at publishers:
3 July 1992
SUMMARY
The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined. A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation. However, unlike its Escherichia coli equivalent, A. tumefuciens recA lacks the upstream ‘SOS box’, suggesting a different mechanism of regulation for this gene.
rand et al. (1989) cloned recA from A. tumefaciens. We have now determined its complete sequence (Fig. 1).
INTRODUCTION
The gene recA plays an important role in both genetic recombination (Mahajan, 1988) and the induction of SOS functions (Witkin, 1976). Several in vitro studies showed that the RecA product promotes many vital reactions. These include annealing of complementary single-stranded (ss) DNAs (Weinstock et al., 1979), assimilation of ssDNA to a homologous region of duplex DNA to produce a D-loop structure (McEntee et al., 1979). proteolytic cleavage of phage repressors (Phizicky and Roberts, 1980) and the /exA gene product (Little et al., 1981). Several interesting properties of A. tumefaciens such as those involved in plant tumorigenesis and its use in genetic engineering are well-known. Therefore, we decided to learn more about the role of the genes responsible for recombination and stability of foreign genes in this bacterium. As a first step towards this goal, Miles et al. (1986) and Far-
Correspondence to: Dr. G.R.K.
Sastry,
Department
of Genetics,
Univer-
sity of Leeds, Leeds LS2 9JT, UK. Tel. (44-532)333098; Fax (44-532)441175. Abbreviations: kilobase
A., Agrohcrcterium; aa, amino acid(s); bp, base pair(s); kb,
or 1000 bp; nt, nucleotide(s);
ribosomc-binding
ORF, open reading frame; RBS,
site; recA. gene encoding
RecA; RecA, recombinase
A.
EXPERIMENTAL
AND DISCUSSION
(a) Salient features In Fig. 1, the nt sequence l-1092 represents the recA coding region. The putative RBS, 5’-AGGT, is underlined and is located 7 bp upstream from the presumed ATG start codon. An A+T-rich interrupted repeat located immediately downstream of the recA coding region may participate in transcription termination or mRNA stabilization. (b) Comparison of sequences A. tumefaciens RecA aa sequence was compared with other known sequences from Rhizobium meliloti (W. Buikama, personal communication), Proteus mirabilis (Akaboshi et al., 1989) Serratia marcescens (Ball et al., 1990), Escherichia coli (Sancar et al., 1980), Pseudomonas aeruginosa (Sano and Kageyama, 1987), Anabaena variabilis (Owttrim and Coleman, 1989) and Synechococcus strain PCC7002 (Murphy et al., 1990). This comparison revealed the conserved nature of the recA gene in A. tumefaciens (Fig. 2); its aa sequence shares 89”/b, 63”,,, 63x, 6396, 62 ;/; , and 59”j0 identity with the above proteins, respec-
134 87 2 4 32 94 62 184 92 274 122 364 152 454 182 544 212 634 242 724 272 814 302 904 332 994 362 1084 363 ,174 1264 1282
Fig. I. Nucleotide asterisks
sequence
of the Agrohtrcrrriwl
denote the stop codons.
The deduced
tww/irc;w.s XI scqucnce
recA gene and flanking
sequences.
1s also shown. The sequences
tively (Fig. 2). Further confirming the conserved nature of the gene across a wide range of bacterial taxa, several regions of strong aa identity have been observed. The RecA proteins from A. tutnqfaciens and E. coli share many regions of homology, including a putative nt-binding site (overscored region in Fig. 2), which is found in a number of ATP-ADP binding proteins (Higgins et al., 1985). Further conserved regions between the A. tumcfaciens and E. colt’ RecA proteins have been identified on the basis of reported analyses of E. coli recA mutants. The recA 1 mutant in E. coli has been found to be deficient in all known functions (Kawashima et al., 1984); in this mutant Gly”” is replaced by an Asp, and Gly I’” is conserved between the A. tumgfaciens and E. colt’ recA proteins; residues Lys6 to Gly3” in Fig. 2 (underscored region) have been identified as the site for ssDNA binding, Ala”’ to Ile’28 as the ‘active site’ for ATP hydrolysis, and GlyZo4 to Gly”’ as the bacteriophage-repressor-recognition region (Ogawa and Ogawa, 1986). The sequences DIAL (aa 48-51, Fig. 2), AEHA (aa 95-111, Fig. 2) and GDS (aa 160-162, Fig. 2) located in the N-terminal region of the A. tumefaciens recA protein share homology with the active site sequences of several serine proteases (Dayhoff, 1972). However, the extent of conservation towards the C-termini of the RecA proteins is poor. This region in E. colt’ is required both for binding ATP to RecA protein (Banks and Sedgwick, 1986) and for regulation of the protease activity of recA product
The putative
arc registered
RBS (Shine Dalgarno
in GenBank
type) IS underscored;
under the accession
numhcr
M3677h.
(Benedict et al., 1988). We recognized the product of the A. tumefbciens C58 recA gene (44 kDa, as was predicted) by its expression in minicells (data not shown); this was done by using a modified procedure of Clark-Curtiss and Curtiss (1983). In spite of the conserved nature of the A. tum
GENE
Publishers
B.V. All rights reserved.
133
0378-l 119/92/$05.00
06720
analysis of the recA gene of Agrobacterium tumefaciens C58
Molecular (Homologous
recombination;
H. Wardhan”, Depurtrnenr.~
M.J. McPhersonb,
nucleotide-binding
Carol A. Harris’,
domain)
Ela Sharmad
and G.R.K.
Sastry”
14‘IGenerics und ” Biochemistr_v and h Biotechnology Unit, University of Leeds. Leeds. UK: and ’ Department of Biochemistry. GIrtso Group Re-
search Ltd., Greenfbrd. Middlesex. Received
SOS regulation;
by T.D. McKnight:
UK
22 August
1991; Accepted:
22 September
1991; Received
at publishers:
3 July 1992
SUMMARY
The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined. A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation. However, unlike its Escherichia coli equivalent, A. tumefuciens recA lacks the upstream ‘SOS box’, suggesting a different mechanism of regulation for this gene.
rand et al. (1989) cloned recA from A. tumefaciens. We have now determined its complete sequence (Fig. 1).
INTRODUCTION
The gene recA plays an important role in both genetic recombination (Mahajan, 1988) and the induction of SOS functions (Witkin, 1976). Several in vitro studies showed that the RecA product promotes many vital reactions. These include annealing of complementary single-stranded (ss) DNAs (Weinstock et al., 1979), assimilation of ssDNA to a homologous region of duplex DNA to produce a D-loop structure (McEntee et al., 1979). proteolytic cleavage of phage repressors (Phizicky and Roberts, 1980) and the /exA gene product (Little et al., 1981). Several interesting properties of A. tumefaciens such as those involved in plant tumorigenesis and its use in genetic engineering are well-known. Therefore, we decided to learn more about the role of the genes responsible for recombination and stability of foreign genes in this bacterium. As a first step towards this goal, Miles et al. (1986) and Far-
Correspondence to: Dr. G.R.K.
Sastry,
Department
of Genetics,
Univer-
sity of Leeds, Leeds LS2 9JT, UK. Tel. (44-532)333098; Fax (44-532)441175. Abbreviations: kilobase
A., Agrohcrcterium; aa, amino acid(s); bp, base pair(s); kb,
or 1000 bp; nt, nucleotide(s);
ribosomc-binding
ORF, open reading frame; RBS,
site; recA. gene encoding
RecA; RecA, recombinase
A.
EXPERIMENTAL
AND DISCUSSION
(a) Salient features In Fig. 1, the nt sequence l-1092 represents the recA coding region. The putative RBS, 5’-AGGT, is underlined and is located 7 bp upstream from the presumed ATG start codon. An A+T-rich interrupted repeat located immediately downstream of the recA coding region may participate in transcription termination or mRNA stabilization. (b) Comparison of sequences A. tumefaciens RecA aa sequence was compared with other known sequences from Rhizobium meliloti (W. Buikama, personal communication), Proteus mirabilis (Akaboshi et al., 1989) Serratia marcescens (Ball et al., 1990), Escherichia coli (Sancar et al., 1980), Pseudomonas aeruginosa (Sano and Kageyama, 1987), Anabaena variabilis (Owttrim and Coleman, 1989) and Synechococcus strain PCC7002 (Murphy et al., 1990). This comparison revealed the conserved nature of the recA gene in A. tumefaciens (Fig. 2); its aa sequence shares 89”/b, 63”,,, 63x, 6396, 62 ;/; , and 59”j0 identity with the above proteins, respec-
134 87 2 4 32 94 62 184 92 274 122 364 152 454 182 544 212 634 242 724 272 814 302 904 332 994 362 1084 363 ,174 1264 1282
Fig. I. Nucleotide asterisks
sequence
of the Agrohtrcrrriwl
denote the stop codons.
The deduced
tww/irc;w.s XI scqucnce
recA gene and flanking
sequences.
1s also shown. The sequences
tively (Fig. 2). Further confirming the conserved nature of the gene across a wide range of bacterial taxa, several regions of strong aa identity have been observed. The RecA proteins from A. tutnqfaciens and E. coli share many regions of homology, including a putative nt-binding site (overscored region in Fig. 2), which is found in a number of ATP-ADP binding proteins (Higgins et al., 1985). Further conserved regions between the A. tumcfaciens and E. colt’ RecA proteins have been identified on the basis of reported analyses of E. coli recA mutants. The recA 1 mutant in E. coli has been found to be deficient in all known functions (Kawashima et al., 1984); in this mutant Gly”” is replaced by an Asp, and Gly I’” is conserved between the A. tumgfaciens and E. colt’ recA proteins; residues Lys6 to Gly3” in Fig. 2 (underscored region) have been identified as the site for ssDNA binding, Ala”’ to Ile’28 as the ‘active site’ for ATP hydrolysis, and GlyZo4 to Gly”’ as the bacteriophage-repressor-recognition region (Ogawa and Ogawa, 1986). The sequences DIAL (aa 48-51, Fig. 2), AEHA (aa 95-111, Fig. 2) and GDS (aa 160-162, Fig. 2) located in the N-terminal region of the A. tumefaciens recA protein share homology with the active site sequences of several serine proteases (Dayhoff, 1972). However, the extent of conservation towards the C-termini of the RecA proteins is poor. This region in E. colt’ is required both for binding ATP to RecA protein (Banks and Sedgwick, 1986) and for regulation of the protease activity of recA product
The putative
arc registered
RBS (Shine Dalgarno
in GenBank
type) IS underscored;
under the accession
numhcr
M3677h.
(Benedict et al., 1988). We recognized the product of the A. tumefbciens C58 recA gene (44 kDa, as was predicted) by its expression in minicells (data not shown); this was done by using a modified procedure of Clark-Curtiss and Curtiss (1983). In spite of the conserved nature of the A. tum
