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Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli
1
Gene, 78 (1989) l-8
Elsevier GEN 02959 Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia
coli (DNA repair; protease activity; protein homology; phage J and Lex repressors; recombination)
Rajkumar S. Ramesar, Valerie Abratt, David R. Woods and Douglas E. Rawlings Department of Microbiology, Universityof Cape Town,Rondebosch,7700 (South Africa) Received
by R.E. Yasbin:
Revised:
19 October
Accepted:
8 August
1988
1988
2 November
1988
SUMMARY
The nucleotide sequence of the recA gene of Thiobacillusferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the 1 pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the I and E. coli LexA repressors.
INTRODUCTION
T. ferrooxidans
is an acidophilic, diazotrophic, autotrophic bacterium that can obtain energy through the oxidation of ferrous iron or reduced inorganic sulfur compounds. It is able to grow in Correspondence ment,
to: Dr. D.E. Rawlings,
University
of Cape
Africa) Tel. (021)650-3261; Abbreviations:
Town,
otide;
aa, amino
sulfonate; ORF,
open
electrophoresis; Delgamo;
acid(s);
0378-l 119/89/$03.50
MC, mito-
MMS, methyl
nt, nucleotide;
oligo, oligodeoxyribonuclePAGE,
pfu, I;
bp, base pair(s); concentration;
reading
SDS, sodium
(A), prophage
Depart-
7700 (South
Fax (021)650-3726.
mycin C; MIC, minimum inhibitory methane
Microbiology
Rondebosch,
frame;
plaque-forming dodecyl
[ 1,designates
sulfate;
polyacrylamide-gel
units;
plasmid-carrier
0 1989 Elsevier
SD,
UV, ultraviolet
Shinelight;
state.
Science Publishers
B.V. (Biomedical
inorganic mining environments where it is used industrially to leach a variety of metals from pyritic ores. Since little is known about the molecular biology of this bacterium, we have investigated the structure of the recA gene and the derived protein of this physiologically unique organism. The RecA protein of E. coli plays an essential role in homologous recombination (Radding, 1982), induction of the SOS response and in initiation of stable DNA replication (Walker, 1984; Kogoma et al., 1985). The recA genes from E. coli (Sancar et al., 1980) and Pseudomonas aeruginosa (San0 and Kageyama, 1987) have been sequenced and specify proteins of 352 and 346 aa, respectively. Several investigators have isolated E. coli recA mutants and correlated changes in the biological properties of the Division)
2
mutant proteins with corresponding changes in the amino acid sequence (Kawashima et al., 1984; Wang and Tessman, 1986). In this way functional domains of the RecA protein have been assigned. Ramesar et al. (1988) reported the cloning and expression of a recA-like gene from ~~~o~x~~~. The T. ferrooxidans RecA protein was found to partially complement defects in DNA repair and homologous recombination in several E. coii recA mut~t strains. We report the sequence of the T. ferrooxidans recA gene and flanking regions. The derived aa sequence of the recA gene product is compared with those of the recA proteins of E. coli and P. ~e~~inosa.
(c) The recA gene activity
RecA protein function was assayed in E. coli HB 101 recA - strains by the resistance of the cells to MMS and UV irradiation as described by Ramesar et al. (1988). The test for transc~ption from the /zp, promoter of the cloning vector pEcoR251 was carried out using the temperature-sensitive ,? repressor coded by gene ~1857, carried on plasmid ~~1857 (Remaut et al., 1981). This plasmid, which is compatible with pEcoR251, was transformed into E. coli HBlOl[pRSRlOO]. ExpressionoftherecAgenewas assayed at both permissive (30°C) and non-permissive temperatures (42” C). (d) Prophage induction
MATERIALS
AND METHODS
(a) Subcioning
and sequencing
Plasmids pRSRlO0 and pRSRlO1 (Ramesar et al., 1988) which contained the cloned T. ferrooxidans recA gene were used as the primary sources of DNA. Standard molecular genetic techniques (Maniatis et al., 1982) were used to subclone DNA fragments into plasmids pUC18, pUC19 (YanischPerron et al., 1985) and Bluescript (Stratagene, California) vectors. DNA for sequencing was prepared by a combination of subcloning from available restriction sites and the construction of ordered deletions using the enzyme BAL 31 (Mishra, 1985). The sequence was determined from both strands by the dideoxy chain-termination method of Sanger et al. (1977). Sequencing reactions were carried out using primers from Bethesda Research Laboratories and a sequencing kit obtained from Amersham Corp. The DNA chains were radiolabelled with [35S]dCTP (400 Ci/mmol) or [ 35S]dATP (410 Ci/mmol) supplied by Amersham Corp.
Lysogens were constructed by cross-streaking cells against high titre /z phage on L-agar (Arber et al., 1983). Survivors were tested for resistance to superinfection and for the ability to release resident prophage. Lysogens were grown to early log phase (A600 = 0.4), centrifuged, washed and resuspended in L-broth with or without 5.0 pg MC/ml. Cells were incubated for 3 h at 37°C in the dark, lysed with chloroform and cell debris was removed by centrifugation. Lysates were assayed for pfu with the indicator E. coli LE392 strain as described by Ramesar et al. (1988). (e) Western blotting
Western blotting from SDS-polyacrylamide gels onto nitrocellulose membranes was done as described previously (Ramesar et al., 1988). Rabbit antiserum prepared against E. coli RecA protein (Goodman et al., 1987) was used to challenge proteins from crude extracts of E. coli and T. ferrooxidans.
(b) Sequence analysis
Nucleotide and derived amino acid sequences were analysed using the DNA tools and Genepro (version 4.1) programmes. Amino acid sequences were analysed and compared using the protein alignment subroutine of the Beckman Microgenie sequence analysis programme. Stem and loop secondary-structure stability was calculated according to Salser et al. (1977).
RESULTS AND DISCUSSION
(a) Nueleotide
sequence of the recA gene
A 1406-bp fragment of DNA containing the recA gene and flanking regions is shown in Fig. 1. The sequence contained a 1038-bp T.~~ooxidan~
3
1 121 1
TTGGCGATTGCTGGGTn;TC~TMC~~GCCGGTAACCCCGCCCGCCCGCTCAGGTAGRATGCCGGAAAAGGTTGTTCGCCGCCGCGGCG GGTCCTGCCGCGCGCCACTACWWGGTGTTTTWtAG‘?ATGGTTACCCCTCGATTCTGWAAGG AAMTATT ___________
GCCCTGTCA CAG ATT GAC AM CAG TTT GGT 12ALSQIDKQPGKGAVMRLGDH'NAIKDIEVYS
229
GGC TCG CTG GGT CTG GAT CTG GCG CTG TGSLGLDLALGVGGLPRGRVVEIYGPESSG
ATG GAT GAA CAG CGC AGC AAG GGC CT-TTCG GCG M D E Q R S X G L S A
AM
GGC
GCC
GTC
ATG
CGT
CTC
GGT GAT
GGT
GTT
GGC
GGA
CTG
CCC
CGG
GGC CGG Gn:
CAT
MC
GCC ATC
GTA
GAG
MG
GAC
ATC
ATC TAC GGG CCG
GAG
GTC
TAC TCC
GM
TCT
TCC
3::
ACC
400 72
A&A ACG ACT CTC ACC CTG CAT GCC ATA GCC AGT TGT CAG GCT GCA GGC GGC ACC GCC GCC TTT ATC GAT GCC GAG CAC GCG CTC GAC CCA K T T L T L H A I A S C Q A A G G T A A F I D A E H A L D P
490
GGC
TAT
GCC
CAC
AAG
CTC
GGC
GTC
GAT
CTG
G
Y
A
H
K
L
G
V
D
L
CTG
GTG
CGC
TCC
GGT
GCC
GTG
GAC
L
V
R
S
G
A
V
D
102 560
132
GM
E
CTCATCGTC L
I
V
AAC
CTC
CTG
N
L
L
ATC
GAC D
I
TCC
S
TCC
CAG
CCT
GAT
ACC
GGC
GAG
CAG
S
Q
P
D
T
G
E
Q
GTG
GCC
GCT
CTG
ACC
CCC
V
A
A
L
T
P
ATC
I
AAA K
GCG A
GM E
GCC A
CTG
ATC
GM
I
L E
ATC TCC CGG AGC MC I S R S N
GM
E GGC
G
ATC
GGT
GCC
GAC
A
D
N
GAG
An:
GGC
GAT
E
M
G
D
I
ATG
670 162
TCC CAC GTC GGT CTG CAG GCG CGT CTG ATG AGT CAG GCT TTG CGC AAC TTA ACC GCC MT S H V G X Q A R L M S Q A L I N L T A N
ACC CTG GTC ATT TTT T L V I F
760 192
ATC AAC CAG ATT CGC ATG AAA ATC GGG GTG ATG TAT GGC AGT CCG GM I N Q I R M K I G V M Y G S P E
;:i
CGC CTT GAT ATC CGC CGC ATC GGC GCG ATC AAA AAG AGC GAC GAA GTG GTA GGT AAC GAT ACC CGC GTC MG RLDIRRIGAIKXSDEVVGNDTRVKVVXNKV
940 252
GCA CCA CCT TTC CGC GAA GCC GAA TTT GCC ATC TAT TAC GGT GAA GGC ATC TCC CGA CTG TCC GAA CTG GTG GAC CTC GGT GTG AAG TTC A P P P R E A E F A I Y Y G E G I S R L S E L V D L G V L P
ACC ACC ACC GGT GGT AAT GCC CTT AAA TTC TAC GCT TCC GTG T T T G G N A L K P Y A S V GTG GTC AAG MT
1030 262
GAC ATC GTC GM D I V E
1120 3l2
CAT CCG GM H P E
1210 342
CAG CGG TCG GCT AGT TGA CGACRGAACGCAGCGATCCCACCGCCTGGCACCTACGGTTCT~CGCGCC~GAGTA~CGCC~AG~~AC~CTGCTCCGTGC~ Q R S A S .
1323
GATGTGACGCAGGGGACGn;CCTGCGCTGGATGCGCTCGCCACGCGCCTGGTCAT
AAG GTC
AAA AGC GGC GCC TGG TAC AGT TAC CAG GGC CAC CGT ATT GGT CAG GGC AAG GAC AAT GCC CGC CAG TAC CTC AAG GTG K S G A W Y S Y Q G H R I G Q G K D N A R Q Y L K V
CTG GCG GCC MT L A A N
ATC GAG CAG CGG ATA CGG GCA GCG GCA GCA GGA CAC CCC CTG GCC TTT GCC GAA GAG GTG GAG AGC CCG I B Q R I R A A A A G H P L A F A E E V E S P
Fig. 1. Nucleotide sequence of the recA gene and flanking regions of T. femoxiduns. Only the strand with the polarity of the mRNA is shown. The deduced amino acid sequence is given below the coding region. Arrows above the sequence indicate sets of tandem or inverted repeats. An SD-like region is indicated by a dashed line under the nucleotide sequence.
ORF which is preceded by a GGAGAAGGAAA sequence 5 bp upstream from the presumptive start codon (nt position 196). This A + G-rich region contains sequences which resemble other SD sequences (Shine and Dalgarno, 1976) and is situated at the correct distance from the ORF to be regarded as a putative SD-like region. The recA genes of both E. coli and P. aeruginosa have clearly identifiable SOS boxes (San0 and Kageyama, 1987) which contain a consensus LexA-binding site (Walker, 1984) within 57 and 41 bp of the ATG start codon, respectively. No SOS box is present in the 196-bp sequence upstream from the T. ferrooxidans gene and there is also no apparent -35, -10 consensus promoter sequence present. The most notable structural features in the nucleotide sequence of the upstream region are two directly repeated 13-bp sequences in tandem (nt 23-48) and a lo-bp complementary inverted repeat sequence (nt 160-169), 3 1 bp from the ATG start codon. Downstream from the recA ORF are two directly repeated 14-bp sequences that have a single bp mismatch. Within the ORF are two complementary inverted 14-bp repeat sequences (nt 1008-1038) which have two single bp
mismatches and are capable of forming a singlestranded stem-and-loop structure (dG = -23.95 kcal/mol). (b) Expression of the cloned recA gene in Escherichiu
coli Since no promoter sequences were apparent in the region immediately upstream from the recA ORF, we investigated the expression of the cloned gene in E. coli. Three independently cloned T. ferrooxidans recA genes (Ramesar et al., 1988) had the recA ORF in the identical orientation, between 2.1 and 2.5 kb downstream from the I pR promoter of the pEcoR251 cloning vector. A plasmid, pRSR105, was constructed in which the 650-bp EcoRI fragment of pRSRlO0 including the il pR promoter was deleted (Fig. 2). E. coli recA strains containing pRSR105 showed markedly reduced levels of resistance to MMS and UV irradiation (Table I). To confirm that the T. ferrooxidans recA gene on pRSRlO0 was transcribed from the I pR promoter, plasmid ~~1857 (Remaut et al., 198 1) which codes for a temperaturesensitive I repressor was transformed into E. coli
4
s 1
pRSR 100
-
E3HS
s
H
.
PE-1
VECTOR
T.
DNA
fernwxfdms
damaging HBlOl
strain (not shown). These two observations
indicated
that the cloned recA gene on pRSRlO0
expressed pRSR 101
;2
from the A pR promoter
?_;
H
77
S
-
the T. ferrooxidans recA
whether
gene was expressed orientation
when
with respect
SK
PlSC r
H
Fig. 2. A map of plasmid gene and the I
pRSRlO0
sites indicated
Sau3A.
Plasmid
pRSR106
an
and
pRSRlO0
of related
is a BglII-Sau3A
EcoRI
deletion
pRSRl07
contain
cloned in opposite
sub-
The restriction
are: B, EglII; E, EcoRI;
pRSRlO1
pRSRlO5
and maps
between the T. ferrooxiduns recA
pR and p,,, vector promoters.
enzyme mid
I
WEXR
clones showing the relationship
H, XindIII;
deletion
of pRSR100. the
orientations
Hind111
S,
and plasPlasmids
fragment
of
into the Bluescript
SK
vector.
HBlOl[pRSRlOO]. At 42°C resistance to MMS and UV of the transformants was the same as that for the strain lacking the temperature-sensitive repressor. At 30°C resistance to these DNATABLE Expression
of the cloned
Thiobacillusferrooxidam recA gene in
and plasmid”
MIC of MMS
UV dose for 10%
(% vW
survival
E. coli HBlOl
0.002
2
E. cob RR1
0.500
80
E. coli HBlOl[pDR1453]
0.400
55
E. co/i HBlOl[pRSRlOO]
0.300
30
E. coli HBlOl[pRSRlOl]
0.300
30
E. coli HBlOl[pRSR105]
0.060
5
E. coli HBlOl[pRSR106]
0.200
20
E. coli HBlOl[pRSR107]
0.080
(s) ’
7.5
a E. coli strains
HB 10 1 and RR1 are rec4 + and recA -, respecpDR1453
and Rupp,
oxidans recA gene. pRSRlO0
carries
the cloned
1978) and pRSRlO0 Plasmids
E. coli recA gene
the cloned
pRSRlOl-107
T. ferro-
are subclones
of
(Fig. 2).
b Determined
by the ability of cells to form colonies
night incubation centrations
A
a
into the Bluescript
recombinant
plasmid
same levels of MMS and UVresistance as pRSRlO0 (Table I). In contrast, when the recA gene was cloned in the opposite orientation (pRSR107) gene expression was reduced to that of pRSR105. These results indicated that the 2.2-kb DNA fragment upstream from the cloned T. ferrooxidans recA gene did not contain a promoter that was efficiently expressed in E. cob. It is not known whether this is because the T. ferrooxidans recA gene has a promoter that is not recognised in E. coli, or whether unlike E. coli and P. aeruginosa its promoter 2.2 kb away.
is located
more
than
at 37°C on Luria agar containing
of MMS.
’ UV irradiation
was at
1 J/m*; s = seconds.
The predicted amino acid sequence of the T. ferrooxidans RecA protein is 346 aa in length. This is the same as that of P. aeruginosa and six residues shorter than the E. coli recA gene product (Sancar
tively. Plasmid (Sancar
(Fig. 2).
promoter, the recA ORF
(c) Comparative analysis of the recA gene products
I
Escherichia coli Strain
plasmid
in the opposite
(pRSR106) in which the cloned recA gene was situated 2.0 kb downstream from the luc promoter of the Bluescript vector, resulted in approximately the
H
I
L-
B
containing
was cloned in both orientations
‘;-
pRSR 107
cloned
to a vector
3.3-kb Hind111 fragment pRSR 106
was
of the cloning
vector. To determine
pRSR 105
to that of the E. coli
agents was reduced
after over-
different con-
et al., 1980). The T. ferrooxidans RecA pro-
tein shares a high degree of homology with both the E. coli (66%) and P. aeruginosa (69%) RecA proteins. A comparison of the derived amino acid sequence between the three completely sequenced recA genes and a partial sequence of the recA gene from the cyanobacterium Synechococcus (Murphy et al., 1987) is shown in Fig. 3. There is a striking degree of homology extending throughout the proteins except for two regions. There is a small region of poor homology at the N terminus (aa residues 30-41 of the T. ferrooxidans protein) and a larger region of limited homology (approx. 40 aa) at the C terminus. The greatest amount of conservation between the three eubacterial RecA proteins is in the region from Ile- 190 to Arg-227. The amino acid sequences of the
5
EC
200
Tf Pa SY
200 199
Fig. 3. A comparison between the amino acid sequences of the RecA proteins of E. coli (EC) (Sancar et al., 1988); T.ferrooxidans (Tf); Synechococcus (Sy) (Murphy et al., 1987). Boxed residues are identical in at least three of the four proteins. Dashes represent breaks in the amino acid sequence introduced to allow maximum alignment.
P. aemginosa (Pa) (Sano and Kageyama, 1987) and the partial sequence of
E. coli and P. aeruginosa recA gene products are per-
fectly conserved within this region and there is only a single aa substitution (E. coli, Phe-203 to T. ferrooxidans, Tyr-203). Of particular interest is the degree of conservation in the regions already identified as functional domains. The effect of mutations in the recA gene on the amino acid sequence and protein activity has been studied by several workers (Table II). Most of the amino acid substitutions which affected the function of the E. coli RecA protein are conserved in the T. ferrooxidans protein. Exceptions are the presence of T. ferrooxidans Ile-I 16 in place of E. coli RecA Cys-116 and several changes in the amino acids associated with elevated levels of protease activity in the absence of DNA-damaging agents. These are E. coli amino acids Ser-25, Thr-39, Ala- 179 and Ghr-184, which have been replaced by Ala-24, Val-39, Thr-179 and Arg-184 in the corresponding positions of the T. ferrooxidans RecA protein. An ATP-binding domain of the E. coli RecA pro-
tein was identified by Knight and McEntee (1985; 1986). These workers isolated a 24-residue peptide (Gln-257 to Lys-280) from a tryptic digest of the RecA protein that was able to bind ATP in solution and identified Tyr-264 as the site of modification using the photoaffinity label B-azidoadenosine 5’-triphosphate. Knight et al. (1988) isolated similar peptides from several members of the Enterobacteriaceae and found that they were highly conserved with respect to their elution properties and their ability to hybridize to oligo probes for this ATPbinding domain. A comparison of the amino acid sequences equivalent to the Gly-257 to Lys-280 peptide between E. coli, T. ferrooxidans and P. aerugihosa indicates that only 11 out of 24 aa residues are conserved between all three of these more distantly related bacteria (Fig. 3). The equivalent amino acid sequence from the cyanobacterium, Synechoccus, is even less well conserved and has the Tyr-264 of the RecA protein E. coli substituted by Phe. The C terminus of the RecA protein has been
6
TABLE II A comparison of amino acid substitutions that affect E.r&etic!ziacoli RecA function with the corresponding aa in ~;“liobucilfusSerroo
( ?‘$ ) and Pseudomonas aerugitma (P. a. ) Amino acid substitution in E. coii”
Function affected
Gly-160 -+ Asp
All
Gly-204 + Ser
cys-116
Ser-25 --f Phe Glu-38 -+ Lys Thr-39 --t Be Glu-158 + Lys Ala- 179 -+ Val Gln- 184 -+ Lys Arg-169 --f Cys Gly-301 --f Ser Gly-301+ Asp
TJC
P.a.
Kawashima et al. (1984)
Gly-160
Gly-160
Defective protease activity
Kawashima et al. (1984)
Gly-204
Gly-203
ATPase
Kuramistu et al. (1984)
Ile-116
Val-11.5
Ala-24 Glu-38 Val-39 Glu-158 Thr-179 Arg-184
Ala-24 Pro-37 Ala-38 Glu-157 Thr-178 Asn-183
in E. colib
Protease constitutive
Wang and Tessman (1986)
Protease constitutive and recombinase deficient
Wang and Tessman (1986)
Arg-169 Gly-301
Arg-168 Gly-300
Kawashima et al. (1984) Wang and Tessman (1986)
Val-246
Val-245
Be-298
Be-297
Val-246 --t Met Be-298 -+ Val
Corresponding amino acids
Reference
Thermosensitivity
a Cys-116 chemically modified by ~-(7-dimethylamino-4-methylcouma~nyl)-m~eimide. b All indicates totaf inactivation of the RecA protein.
found to affect protease activity. Evidence for this conclusion is the E. coli recA5327 mutant protein which lacks the 25 C-terminal aa has a high level protease activity in the absence of DNA damage (Ogawa and Ogawa, 1986). The amino acid sequences of this region are poorly conserved between all three eubacterial RecA proteins (Fig. 3).
2 after exposure to MC in a recA 1 lysogen is shown in Table III. Resident n prophage was neither induced spontaneously nor after DNA damage in E. coli HBlOl recA cells. Spontaneous prophage induction in E. coli /1lysogens was st~ulat~ by TABLE III Effect of the cloned recA + gene on the induction of prophage d
(d) Proteolytic activity of the Thiobacillus j&moxidans RecA protein
We investigated whether the T. ~rrooxidun~ RecA protein was proteol~ic~ly active toward the i. and LexA repressors despite changes in several amino acids that are known to affect E. coli RecA protease activity. The RecA protein may not be a true protease but only an allosteric affector of 1 and LexA repressor autodigestion (Little, 1984). Induction of phage
Lysogen a
E. coii HBlOl(I) E. co& RRi(3)
E. coli HBlOl[pDR1453](1) E. coli HBlOl[pRSR100](1)
Phage produced (pfu/ml) b Spontaneous
MC induced
18 7.5 x 105 8.6 x IO5 2.0 x 106
26 1.96 x 10’ 1.65 x lo7 1.04 x 10’
a Strain E. coli HBlOl(I) is recA -. b See MATERIALS AND METHODS, section d.
1
&*p+
B
A-
+
D
C
-
+
-
+
as a result
E -
+
-
of DNA
in both E. coli and
damage
T. ferrooxidans as well as the absence binding
site in the nucleotide
of a LexA-
sequence
from the recA ORF, indicates
upstream
that unlike E. coli, the
T. ferrooxidans recA gene may not be regulated E. coli LexA-like protein. 30-
(e) Conclusions
Fig. 4. Western blots of crude protein extracts (+ ) and
uninduced
HBlOl [pRSRlOO]
(- )
T. ferrooxidans
from MC-induced (lane
A),
kDa) are indicated.
Cultures
(lane E). M, markers prepared
after 40 min for
E. coli cells and 90 min for T. ferrooxiduns cells. Western carried
both the (pRSR100)
out section
as
(in
of cells (Aso = 0.4) were treated
with (5 ng MC/ml) and protein extracts
METHODS,
E. coli
(lane B), E. coli RR1 (lane C), E. coli HBlOl
(lane D), and E. coli JK696[pRSRlOO]
was
by a
described
in
MATERIALS
blotting AND
e.
cloned T. ferrooxidans recA and the cloned E. coli recA
gene gene
(pDR1453) (Sancar and Rupp, 1979) to levels approximately equal to those of the recA+ E. coli RR1 cells. MC increased the production of phage 1 26-fold in E. coli RRl, 19-fold in E. coli HBlOl[pDR1453](1) and five-fold in E. coli HBlOl[pRSR100](1) lysogens. The ability of the T. ferrooxidans RecA protein to cleave the LexA repressor was shown using Westernblot experiments (Fig. 4). E. coli HBlOl produces a defective RecA protein that unlike the RecA protein produced by the E. coli RR1 strain has no protease activity and is not induced by DNA damage (lanes C and D). Induction of the defective E. coli HBlOl RecA protein as a result of DNA damage was detected in the presence of the T. ferrooxidans RecA protein (lane B). The most likely reason for this increase is that the E. coli LexA repressor was inactivated by the T. ferrooxidans RecA protein. An increase in the quantity of T. ferrooxidans RecA protein as a result of DNA damage was not detectable in protein preparations from T. ferrooxidans cells (lane A) or from the E. coli JK696 recA strain containing the cloned T. ferrooxidans gene (lane E). E. coli JK696 is a recA deletion strain which lacks a RecA protein and does not partially obscure the T. ferrooxidans RecA protein. These results indicated that DNA damage enhanced the proteolytic activity of the T. ferrooxidans RecA protein toward the Iz and LexA repressors. The lack of induction of the T. ferrooxidans RecA protein
The cloned ciently
T. ferrooxidans recA gene is not effr-
expressed
in E. coli, from the DNA
from the recA ORF.
upstream
196-bp region of DNA immediately the recA ORF was apparent
2.2 kb
No SOS box in the upstream
from
and no induction
of the
cloned T. ferrooxidans recA gene could be detected after DNA damage in E. coli. Likewise, no induction of the RecA protein in T. ferrooxidans was detected and it would be interesting to establish whether the expression of the recA gene in T. ferrooxidans is regulated by a LexA-like protein. The cloned gene was, however, expressed from either the pEcoR25 1 il pR or the pUC19 lac vector promoters. Despite changes in several amino acids that have previously been found to affect E. coli RecA protease activity, the T. ferrooxidans RecA protein has retained DNAdamage-induced proteolytic activity toward both A and the E. coli LexA repressors. These amino acid substitutions did not affect the recombinase function of the T. ferrooxidans RecA protein in E. coli (Ramesar
et al., 1988).
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Harbor, Goodman,
guide to lambda
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In Hendrix,
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R.A. (Eds.),
Laboratory,
Cold Spring
NY, 1983, pp. 381-395. H.J.L., Parker, J.K., Southern,
Cloning and expression from Bacteroides Kawashima, domains
J.A. and Woods, D.R.:
in Escherichia coli of a red-like
gene
frugilti.Gene 58 (1987) 265-271.
H., Horii, T., Ogawa,
T. and Ogawa,
ofEscherichia coliRecA
H.: Functional
protein deduced
from muta-
tional sites in the gene. Mol. Gen. Genet. 193 (1984) 288-292. Knight, K.L. and McEntee,
K.: Tyrosine-264
in the RecA protein
from Escherichiu coli is the site of modification affinity
label
8-azidoadenosine
by the photo-
5’-triphosphate.
J. Biol.
Chem. 260 (1985) 10185-10191. Knight,
K.L.
24-residue
and
McEntee,
K.:
Nucleotide
peptide from the RecA protein
Proc. Natl. Acad.
binding
by
a
of Escherichia cob.
Sci. USA 83 (1986) 9289-9293.
8 Knight, K.L., Hess, R.M. and McEntee, K.: Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Envinia carotovora, Shigelia jlexnen',and Eschenkhia coli K-12 and B/r. J. Bacterial. 170 (1988) 2427-2432. Kogoma, T., Skarstad, K., Boye, E., von Meyenburg, K. and Steen, H.B.: RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12. J. Bacterial. 163 (1985) 439-444. Kuramitsu, S., Hamaguchi, K., Tachibana, H., Horii, T., Ogawa, T. and Ogawa, H.: Cysteinyl residues ofEscherichia coli RecA protein. Biochemistry 23 (1984) 2363-2367. Little, J.W.: Autodigestion of IexA and phage 1 repressors. Proc. Natl. Acad. Sci. USA 81 (1984) 1375-1379. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Mishra, T.: A new strategy to create ordered deletions for rapid DNA sequencing. Gene 34 (1985) 263-268. Murphy, R.A., Bryant, D.A., Porter, R.D. and Tandeau de Marsac, N.: Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002. J. Bacterial. 169 (1987) 2739-2747. Ogawa, H. and Ogawa, T.: General recombination: functions and structure of RecA protein. Adv. Biophys. 21 (1986) 135-148. Radding, C.M.: Homologous pairing and strand exchange in genetic recombination. Annu. Rev. Genet. 16 (1982) 405-437. Ramesar, R.S., Woods, D.R. and Rawlings, D.E.: Cloning and expression in Escherichia coli of a recA-like gene from Thiobacillusferrooxidans. J. Gen. Microbial. 134 (1988) 1141-l 146. Remaut, E., Stanssens, P. and Fiers, W.: Plasmid vectors for high-efficiency expression controlled by the pL promoter of coliphage lambda. Gene 15 (1981) 81-93.
Salser, W.: Globin mRNA sequences: analysis of base pairing and evolutionary implications. Cold Spring Harbor Symp. Quant. Biol. 42 (1977) 985-1002. Sancar, A. and Rupp, W.D.: Physical map of the recA gene. Proc. Natl. Acad. Sci. USA 76 (1979) 3144-3148. Sancar, A., Stachelek, C., Koningsberg, W. and Rupp, W.D.: Sequences ofthe recA gene and protein. Proc. Natl. Acad. Sci. USA 77 (1980) 2611-2615. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Sane, Y. and Kageyama, M.: The sequence and function of the rec4 gene and its protein in Pseudomonas aerugiraosa PAO. Mol. Gen. Genet. 208 (1987) 412-419. Shine, J. and Dalgarno, L.: Determinant of cistron specificity in bacterial ribosomes. Nature 254 (1976) 34-38. Walker, G.C.: Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbial. Rev. 48 (1984) 60-93. Wang, W.-B. and Tessman, E.S.: Evidence that the recA441 (rif) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity. J. Bacterial. 163 (1985) 407-409. Wang, W.-B. and Tessman, E.S.: Location of functional regions of the Eschenkhia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants. J. Bacterial. 168 (1986) 901-910. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33 (1985) 103-119.
Gene, 78 (1989) l-8
Elsevier GEN 02959 Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia
coli (DNA repair; protease activity; protein homology; phage J and Lex repressors; recombination)
Rajkumar S. Ramesar, Valerie Abratt, David R. Woods and Douglas E. Rawlings Department of Microbiology, Universityof Cape Town,Rondebosch,7700 (South Africa) Received
by R.E. Yasbin:
Revised:
19 October
Accepted:
8 August
1988
1988
2 November
1988
SUMMARY
The nucleotide sequence of the recA gene of Thiobacillusferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the 1 pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the I and E. coli LexA repressors.
INTRODUCTION
T. ferrooxidans
is an acidophilic, diazotrophic, autotrophic bacterium that can obtain energy through the oxidation of ferrous iron or reduced inorganic sulfur compounds. It is able to grow in Correspondence ment,
to: Dr. D.E. Rawlings,
University
of Cape
Africa) Tel. (021)650-3261; Abbreviations:
Town,
otide;
aa, amino
sulfonate; ORF,
open
electrophoresis; Delgamo;
acid(s);
0378-l 119/89/$03.50
MC, mito-
MMS, methyl
nt, nucleotide;
oligo, oligodeoxyribonuclePAGE,
pfu, I;
bp, base pair(s); concentration;
reading
SDS, sodium
(A), prophage
Depart-
7700 (South
Fax (021)650-3726.
mycin C; MIC, minimum inhibitory methane
Microbiology
Rondebosch,
frame;
plaque-forming dodecyl
[ 1,designates
sulfate;
polyacrylamide-gel
units;
plasmid-carrier
0 1989 Elsevier
SD,
UV, ultraviolet
Shinelight;
state.
Science Publishers
B.V. (Biomedical
inorganic mining environments where it is used industrially to leach a variety of metals from pyritic ores. Since little is known about the molecular biology of this bacterium, we have investigated the structure of the recA gene and the derived protein of this physiologically unique organism. The RecA protein of E. coli plays an essential role in homologous recombination (Radding, 1982), induction of the SOS response and in initiation of stable DNA replication (Walker, 1984; Kogoma et al., 1985). The recA genes from E. coli (Sancar et al., 1980) and Pseudomonas aeruginosa (San0 and Kageyama, 1987) have been sequenced and specify proteins of 352 and 346 aa, respectively. Several investigators have isolated E. coli recA mutants and correlated changes in the biological properties of the Division)
2
mutant proteins with corresponding changes in the amino acid sequence (Kawashima et al., 1984; Wang and Tessman, 1986). In this way functional domains of the RecA protein have been assigned. Ramesar et al. (1988) reported the cloning and expression of a recA-like gene from ~~~o~x~~~. The T. ferrooxidans RecA protein was found to partially complement defects in DNA repair and homologous recombination in several E. coii recA mut~t strains. We report the sequence of the T. ferrooxidans recA gene and flanking regions. The derived aa sequence of the recA gene product is compared with those of the recA proteins of E. coli and P. ~e~~inosa.
(c) The recA gene activity
RecA protein function was assayed in E. coli HB 101 recA - strains by the resistance of the cells to MMS and UV irradiation as described by Ramesar et al. (1988). The test for transc~ption from the /zp, promoter of the cloning vector pEcoR251 was carried out using the temperature-sensitive ,? repressor coded by gene ~1857, carried on plasmid ~~1857 (Remaut et al., 1981). This plasmid, which is compatible with pEcoR251, was transformed into E. coli HBlOl[pRSRlOO]. ExpressionoftherecAgenewas assayed at both permissive (30°C) and non-permissive temperatures (42” C). (d) Prophage induction
MATERIALS
AND METHODS
(a) Subcioning
and sequencing
Plasmids pRSRlO0 and pRSRlO1 (Ramesar et al., 1988) which contained the cloned T. ferrooxidans recA gene were used as the primary sources of DNA. Standard molecular genetic techniques (Maniatis et al., 1982) were used to subclone DNA fragments into plasmids pUC18, pUC19 (YanischPerron et al., 1985) and Bluescript (Stratagene, California) vectors. DNA for sequencing was prepared by a combination of subcloning from available restriction sites and the construction of ordered deletions using the enzyme BAL 31 (Mishra, 1985). The sequence was determined from both strands by the dideoxy chain-termination method of Sanger et al. (1977). Sequencing reactions were carried out using primers from Bethesda Research Laboratories and a sequencing kit obtained from Amersham Corp. The DNA chains were radiolabelled with [35S]dCTP (400 Ci/mmol) or [ 35S]dATP (410 Ci/mmol) supplied by Amersham Corp.
Lysogens were constructed by cross-streaking cells against high titre /z phage on L-agar (Arber et al., 1983). Survivors were tested for resistance to superinfection and for the ability to release resident prophage. Lysogens were grown to early log phase (A600 = 0.4), centrifuged, washed and resuspended in L-broth with or without 5.0 pg MC/ml. Cells were incubated for 3 h at 37°C in the dark, lysed with chloroform and cell debris was removed by centrifugation. Lysates were assayed for pfu with the indicator E. coli LE392 strain as described by Ramesar et al. (1988). (e) Western blotting
Western blotting from SDS-polyacrylamide gels onto nitrocellulose membranes was done as described previously (Ramesar et al., 1988). Rabbit antiserum prepared against E. coli RecA protein (Goodman et al., 1987) was used to challenge proteins from crude extracts of E. coli and T. ferrooxidans.
(b) Sequence analysis
Nucleotide and derived amino acid sequences were analysed using the DNA tools and Genepro (version 4.1) programmes. Amino acid sequences were analysed and compared using the protein alignment subroutine of the Beckman Microgenie sequence analysis programme. Stem and loop secondary-structure stability was calculated according to Salser et al. (1977).
RESULTS AND DISCUSSION
(a) Nueleotide
sequence of the recA gene
A 1406-bp fragment of DNA containing the recA gene and flanking regions is shown in Fig. 1. The sequence contained a 1038-bp T.~~ooxidan~
3
1 121 1
TTGGCGATTGCTGGGTn;TC~TMC~~GCCGGTAACCCCGCCCGCCCGCTCAGGTAGRATGCCGGAAAAGGTTGTTCGCCGCCGCGGCG GGTCCTGCCGCGCGCCACTACWWGGTGTTTTWtAG‘?ATGGTTACCCCTCGATTCTGWAAGG AAMTATT ___________
GCCCTGTCA CAG ATT GAC AM CAG TTT GGT 12ALSQIDKQPGKGAVMRLGDH'NAIKDIEVYS
229
GGC TCG CTG GGT CTG GAT CTG GCG CTG TGSLGLDLALGVGGLPRGRVVEIYGPESSG
ATG GAT GAA CAG CGC AGC AAG GGC CT-TTCG GCG M D E Q R S X G L S A
AM
GGC
GCC
GTC
ATG
CGT
CTC
GGT GAT
GGT
GTT
GGC
GGA
CTG
CCC
CGG
GGC CGG Gn:
CAT
MC
GCC ATC
GTA
GAG
MG
GAC
ATC
ATC TAC GGG CCG
GAG
GTC
TAC TCC
GM
TCT
TCC
3::
ACC
400 72
A&A ACG ACT CTC ACC CTG CAT GCC ATA GCC AGT TGT CAG GCT GCA GGC GGC ACC GCC GCC TTT ATC GAT GCC GAG CAC GCG CTC GAC CCA K T T L T L H A I A S C Q A A G G T A A F I D A E H A L D P
490
GGC
TAT
GCC
CAC
AAG
CTC
GGC
GTC
GAT
CTG
G
Y
A
H
K
L
G
V
D
L
CTG
GTG
CGC
TCC
GGT
GCC
GTG
GAC
L
V
R
S
G
A
V
D
102 560
132
GM
E
CTCATCGTC L
I
V
AAC
CTC
CTG
N
L
L
ATC
GAC D
I
TCC
S
TCC
CAG
CCT
GAT
ACC
GGC
GAG
CAG
S
Q
P
D
T
G
E
Q
GTG
GCC
GCT
CTG
ACC
CCC
V
A
A
L
T
P
ATC
I
AAA K
GCG A
GM E
GCC A
CTG
ATC
GM
I
L E
ATC TCC CGG AGC MC I S R S N
GM
E GGC
G
ATC
GGT
GCC
GAC
A
D
N
GAG
An:
GGC
GAT
E
M
G
D
I
ATG
670 162
TCC CAC GTC GGT CTG CAG GCG CGT CTG ATG AGT CAG GCT TTG CGC AAC TTA ACC GCC MT S H V G X Q A R L M S Q A L I N L T A N
ACC CTG GTC ATT TTT T L V I F
760 192
ATC AAC CAG ATT CGC ATG AAA ATC GGG GTG ATG TAT GGC AGT CCG GM I N Q I R M K I G V M Y G S P E
;:i
CGC CTT GAT ATC CGC CGC ATC GGC GCG ATC AAA AAG AGC GAC GAA GTG GTA GGT AAC GAT ACC CGC GTC MG RLDIRRIGAIKXSDEVVGNDTRVKVVXNKV
940 252
GCA CCA CCT TTC CGC GAA GCC GAA TTT GCC ATC TAT TAC GGT GAA GGC ATC TCC CGA CTG TCC GAA CTG GTG GAC CTC GGT GTG AAG TTC A P P P R E A E F A I Y Y G E G I S R L S E L V D L G V L P
ACC ACC ACC GGT GGT AAT GCC CTT AAA TTC TAC GCT TCC GTG T T T G G N A L K P Y A S V GTG GTC AAG MT
1030 262
GAC ATC GTC GM D I V E
1120 3l2
CAT CCG GM H P E
1210 342
CAG CGG TCG GCT AGT TGA CGACRGAACGCAGCGATCCCACCGCCTGGCACCTACGGTTCT~CGCGCC~GAGTA~CGCC~AG~~AC~CTGCTCCGTGC~ Q R S A S .
1323
GATGTGACGCAGGGGACGn;CCTGCGCTGGATGCGCTCGCCACGCGCCTGGTCAT
AAG GTC
AAA AGC GGC GCC TGG TAC AGT TAC CAG GGC CAC CGT ATT GGT CAG GGC AAG GAC AAT GCC CGC CAG TAC CTC AAG GTG K S G A W Y S Y Q G H R I G Q G K D N A R Q Y L K V
CTG GCG GCC MT L A A N
ATC GAG CAG CGG ATA CGG GCA GCG GCA GCA GGA CAC CCC CTG GCC TTT GCC GAA GAG GTG GAG AGC CCG I B Q R I R A A A A G H P L A F A E E V E S P
Fig. 1. Nucleotide sequence of the recA gene and flanking regions of T. femoxiduns. Only the strand with the polarity of the mRNA is shown. The deduced amino acid sequence is given below the coding region. Arrows above the sequence indicate sets of tandem or inverted repeats. An SD-like region is indicated by a dashed line under the nucleotide sequence.
ORF which is preceded by a GGAGAAGGAAA sequence 5 bp upstream from the presumptive start codon (nt position 196). This A + G-rich region contains sequences which resemble other SD sequences (Shine and Dalgarno, 1976) and is situated at the correct distance from the ORF to be regarded as a putative SD-like region. The recA genes of both E. coli and P. aeruginosa have clearly identifiable SOS boxes (San0 and Kageyama, 1987) which contain a consensus LexA-binding site (Walker, 1984) within 57 and 41 bp of the ATG start codon, respectively. No SOS box is present in the 196-bp sequence upstream from the T. ferrooxidans gene and there is also no apparent -35, -10 consensus promoter sequence present. The most notable structural features in the nucleotide sequence of the upstream region are two directly repeated 13-bp sequences in tandem (nt 23-48) and a lo-bp complementary inverted repeat sequence (nt 160-169), 3 1 bp from the ATG start codon. Downstream from the recA ORF are two directly repeated 14-bp sequences that have a single bp mismatch. Within the ORF are two complementary inverted 14-bp repeat sequences (nt 1008-1038) which have two single bp
mismatches and are capable of forming a singlestranded stem-and-loop structure (dG = -23.95 kcal/mol). (b) Expression of the cloned recA gene in Escherichiu
coli Since no promoter sequences were apparent in the region immediately upstream from the recA ORF, we investigated the expression of the cloned gene in E. coli. Three independently cloned T. ferrooxidans recA genes (Ramesar et al., 1988) had the recA ORF in the identical orientation, between 2.1 and 2.5 kb downstream from the I pR promoter of the pEcoR251 cloning vector. A plasmid, pRSR105, was constructed in which the 650-bp EcoRI fragment of pRSRlO0 including the il pR promoter was deleted (Fig. 2). E. coli recA strains containing pRSR105 showed markedly reduced levels of resistance to MMS and UV irradiation (Table I). To confirm that the T. ferrooxidans recA gene on pRSRlO0 was transcribed from the I pR promoter, plasmid ~~1857 (Remaut et al., 198 1) which codes for a temperaturesensitive I repressor was transformed into E. coli
4
s 1
pRSR 100
-
E3HS
s
H
.
PE-1
VECTOR
T.
DNA
fernwxfdms
damaging HBlOl
strain (not shown). These two observations
indicated
that the cloned recA gene on pRSRlO0
expressed pRSR 101
;2
from the A pR promoter
?_;
H
77
S
-
the T. ferrooxidans recA
whether
gene was expressed orientation
when
with respect
SK
PlSC r
H
Fig. 2. A map of plasmid gene and the I
pRSRlO0
sites indicated
Sau3A.
Plasmid
pRSR106
an
and
pRSRlO0
of related
is a BglII-Sau3A
EcoRI
deletion
pRSRl07
contain
cloned in opposite
sub-
The restriction
are: B, EglII; E, EcoRI;
pRSRlO1
pRSRlO5
and maps
between the T. ferrooxiduns recA
pR and p,,, vector promoters.
enzyme mid
I
WEXR
clones showing the relationship
H, XindIII;
deletion
of pRSR100. the
orientations
Hind111
S,
and plasPlasmids
fragment
of
into the Bluescript
SK
vector.
HBlOl[pRSRlOO]. At 42°C resistance to MMS and UV of the transformants was the same as that for the strain lacking the temperature-sensitive repressor. At 30°C resistance to these DNATABLE Expression
of the cloned
Thiobacillusferrooxidam recA gene in
and plasmid”
MIC of MMS
UV dose for 10%
(% vW
survival
E. coli HBlOl
0.002
2
E. cob RR1
0.500
80
E. coli HBlOl[pDR1453]
0.400
55
E. co/i HBlOl[pRSRlOO]
0.300
30
E. coli HBlOl[pRSRlOl]
0.300
30
E. coli HBlOl[pRSR105]
0.060
5
E. coli HBlOl[pRSR106]
0.200
20
E. coli HBlOl[pRSR107]
0.080
(s) ’
7.5
a E. coli strains
HB 10 1 and RR1 are rec4 + and recA -, respecpDR1453
and Rupp,
oxidans recA gene. pRSRlO0
carries
the cloned
1978) and pRSRlO0 Plasmids
E. coli recA gene
the cloned
pRSRlOl-107
T. ferro-
are subclones
of
(Fig. 2).
b Determined
by the ability of cells to form colonies
night incubation centrations
A
a
into the Bluescript
recombinant
plasmid
same levels of MMS and UVresistance as pRSRlO0 (Table I). In contrast, when the recA gene was cloned in the opposite orientation (pRSR107) gene expression was reduced to that of pRSR105. These results indicated that the 2.2-kb DNA fragment upstream from the cloned T. ferrooxidans recA gene did not contain a promoter that was efficiently expressed in E. cob. It is not known whether this is because the T. ferrooxidans recA gene has a promoter that is not recognised in E. coli, or whether unlike E. coli and P. aeruginosa its promoter 2.2 kb away.
is located
more
than
at 37°C on Luria agar containing
of MMS.
’ UV irradiation
was at
1 J/m*; s = seconds.
The predicted amino acid sequence of the T. ferrooxidans RecA protein is 346 aa in length. This is the same as that of P. aeruginosa and six residues shorter than the E. coli recA gene product (Sancar
tively. Plasmid (Sancar
(Fig. 2).
promoter, the recA ORF
(c) Comparative analysis of the recA gene products
I
Escherichia coli Strain
plasmid
in the opposite
(pRSR106) in which the cloned recA gene was situated 2.0 kb downstream from the luc promoter of the Bluescript vector, resulted in approximately the
H
I
L-
B
containing
was cloned in both orientations
‘;-
pRSR 107
cloned
to a vector
3.3-kb Hind111 fragment pRSR 106
was
of the cloning
vector. To determine
pRSR 105
to that of the E. coli
agents was reduced
after over-
different con-
et al., 1980). The T. ferrooxidans RecA pro-
tein shares a high degree of homology with both the E. coli (66%) and P. aeruginosa (69%) RecA proteins. A comparison of the derived amino acid sequence between the three completely sequenced recA genes and a partial sequence of the recA gene from the cyanobacterium Synechococcus (Murphy et al., 1987) is shown in Fig. 3. There is a striking degree of homology extending throughout the proteins except for two regions. There is a small region of poor homology at the N terminus (aa residues 30-41 of the T. ferrooxidans protein) and a larger region of limited homology (approx. 40 aa) at the C terminus. The greatest amount of conservation between the three eubacterial RecA proteins is in the region from Ile- 190 to Arg-227. The amino acid sequences of the
5
EC
200
Tf Pa SY
200 199
Fig. 3. A comparison between the amino acid sequences of the RecA proteins of E. coli (EC) (Sancar et al., 1988); T.ferrooxidans (Tf); Synechococcus (Sy) (Murphy et al., 1987). Boxed residues are identical in at least three of the four proteins. Dashes represent breaks in the amino acid sequence introduced to allow maximum alignment.
P. aemginosa (Pa) (Sano and Kageyama, 1987) and the partial sequence of
E. coli and P. aeruginosa recA gene products are per-
fectly conserved within this region and there is only a single aa substitution (E. coli, Phe-203 to T. ferrooxidans, Tyr-203). Of particular interest is the degree of conservation in the regions already identified as functional domains. The effect of mutations in the recA gene on the amino acid sequence and protein activity has been studied by several workers (Table II). Most of the amino acid substitutions which affected the function of the E. coli RecA protein are conserved in the T. ferrooxidans protein. Exceptions are the presence of T. ferrooxidans Ile-I 16 in place of E. coli RecA Cys-116 and several changes in the amino acids associated with elevated levels of protease activity in the absence of DNA-damaging agents. These are E. coli amino acids Ser-25, Thr-39, Ala- 179 and Ghr-184, which have been replaced by Ala-24, Val-39, Thr-179 and Arg-184 in the corresponding positions of the T. ferrooxidans RecA protein. An ATP-binding domain of the E. coli RecA pro-
tein was identified by Knight and McEntee (1985; 1986). These workers isolated a 24-residue peptide (Gln-257 to Lys-280) from a tryptic digest of the RecA protein that was able to bind ATP in solution and identified Tyr-264 as the site of modification using the photoaffinity label B-azidoadenosine 5’-triphosphate. Knight et al. (1988) isolated similar peptides from several members of the Enterobacteriaceae and found that they were highly conserved with respect to their elution properties and their ability to hybridize to oligo probes for this ATPbinding domain. A comparison of the amino acid sequences equivalent to the Gly-257 to Lys-280 peptide between E. coli, T. ferrooxidans and P. aerugihosa indicates that only 11 out of 24 aa residues are conserved between all three of these more distantly related bacteria (Fig. 3). The equivalent amino acid sequence from the cyanobacterium, Synechoccus, is even less well conserved and has the Tyr-264 of the RecA protein E. coli substituted by Phe. The C terminus of the RecA protein has been
6
TABLE II A comparison of amino acid substitutions that affect E.r&etic!ziacoli RecA function with the corresponding aa in ~;“liobucilfusSerroo
( ?‘$ ) and Pseudomonas aerugitma (P. a. ) Amino acid substitution in E. coii”
Function affected
Gly-160 -+ Asp
All
Gly-204 + Ser
cys-116
Ser-25 --f Phe Glu-38 -+ Lys Thr-39 --t Be Glu-158 + Lys Ala- 179 -+ Val Gln- 184 -+ Lys Arg-169 --f Cys Gly-301 --f Ser Gly-301+ Asp
TJC
P.a.
Kawashima et al. (1984)
Gly-160
Gly-160
Defective protease activity
Kawashima et al. (1984)
Gly-204
Gly-203
ATPase
Kuramistu et al. (1984)
Ile-116
Val-11.5
Ala-24 Glu-38 Val-39 Glu-158 Thr-179 Arg-184
Ala-24 Pro-37 Ala-38 Glu-157 Thr-178 Asn-183
in E. colib
Protease constitutive
Wang and Tessman (1986)
Protease constitutive and recombinase deficient
Wang and Tessman (1986)
Arg-169 Gly-301
Arg-168 Gly-300
Kawashima et al. (1984) Wang and Tessman (1986)
Val-246
Val-245
Be-298
Be-297
Val-246 --t Met Be-298 -+ Val
Corresponding amino acids
Reference
Thermosensitivity
a Cys-116 chemically modified by ~-(7-dimethylamino-4-methylcouma~nyl)-m~eimide. b All indicates totaf inactivation of the RecA protein.
found to affect protease activity. Evidence for this conclusion is the E. coli recA5327 mutant protein which lacks the 25 C-terminal aa has a high level protease activity in the absence of DNA damage (Ogawa and Ogawa, 1986). The amino acid sequences of this region are poorly conserved between all three eubacterial RecA proteins (Fig. 3).
2 after exposure to MC in a recA 1 lysogen is shown in Table III. Resident n prophage was neither induced spontaneously nor after DNA damage in E. coli HBlOl recA cells. Spontaneous prophage induction in E. coli /1lysogens was st~ulat~ by TABLE III Effect of the cloned recA + gene on the induction of prophage d
(d) Proteolytic activity of the Thiobacillus j&moxidans RecA protein
We investigated whether the T. ~rrooxidun~ RecA protein was proteol~ic~ly active toward the i. and LexA repressors despite changes in several amino acids that are known to affect E. coli RecA protease activity. The RecA protein may not be a true protease but only an allosteric affector of 1 and LexA repressor autodigestion (Little, 1984). Induction of phage
Lysogen a
E. coii HBlOl(I) E. co& RRi(3)
E. coli HBlOl[pDR1453](1) E. coli HBlOl[pRSR100](1)
Phage produced (pfu/ml) b Spontaneous
MC induced
18 7.5 x 105 8.6 x IO5 2.0 x 106
26 1.96 x 10’ 1.65 x lo7 1.04 x 10’
a Strain E. coli HBlOl(I) is recA -. b See MATERIALS AND METHODS, section d.
1
&*p+
B
A-
+
D
C
-
+
-
+
as a result
E -
+
-
of DNA
in both E. coli and
damage
T. ferrooxidans as well as the absence binding
site in the nucleotide
of a LexA-
sequence
from the recA ORF, indicates
upstream
that unlike E. coli, the
T. ferrooxidans recA gene may not be regulated E. coli LexA-like protein. 30-
(e) Conclusions
Fig. 4. Western blots of crude protein extracts (+ ) and
uninduced
HBlOl [pRSRlOO]
(- )
T. ferrooxidans
from MC-induced (lane
A),
kDa) are indicated.
Cultures
(lane E). M, markers prepared
after 40 min for
E. coli cells and 90 min for T. ferrooxiduns cells. Western carried
both the (pRSR100)
out section
as
(in
of cells (Aso = 0.4) were treated
with (5 ng MC/ml) and protein extracts
METHODS,
E. coli
(lane B), E. coli RR1 (lane C), E. coli HBlOl
(lane D), and E. coli JK696[pRSRlOO]
was
by a
described
in
MATERIALS
blotting AND
e.
cloned T. ferrooxidans recA and the cloned E. coli recA
gene gene
(pDR1453) (Sancar and Rupp, 1979) to levels approximately equal to those of the recA+ E. coli RR1 cells. MC increased the production of phage 1 26-fold in E. coli RRl, 19-fold in E. coli HBlOl[pDR1453](1) and five-fold in E. coli HBlOl[pRSR100](1) lysogens. The ability of the T. ferrooxidans RecA protein to cleave the LexA repressor was shown using Westernblot experiments (Fig. 4). E. coli HBlOl produces a defective RecA protein that unlike the RecA protein produced by the E. coli RR1 strain has no protease activity and is not induced by DNA damage (lanes C and D). Induction of the defective E. coli HBlOl RecA protein as a result of DNA damage was detected in the presence of the T. ferrooxidans RecA protein (lane B). The most likely reason for this increase is that the E. coli LexA repressor was inactivated by the T. ferrooxidans RecA protein. An increase in the quantity of T. ferrooxidans RecA protein as a result of DNA damage was not detectable in protein preparations from T. ferrooxidans cells (lane A) or from the E. coli JK696 recA strain containing the cloned T. ferrooxidans gene (lane E). E. coli JK696 is a recA deletion strain which lacks a RecA protein and does not partially obscure the T. ferrooxidans RecA protein. These results indicated that DNA damage enhanced the proteolytic activity of the T. ferrooxidans RecA protein toward the Iz and LexA repressors. The lack of induction of the T. ferrooxidans RecA protein
The cloned ciently
T. ferrooxidans recA gene is not effr-
expressed
in E. coli, from the DNA
from the recA ORF.
upstream
196-bp region of DNA immediately the recA ORF was apparent
2.2 kb
No SOS box in the upstream
from
and no induction
of the
cloned T. ferrooxidans recA gene could be detected after DNA damage in E. coli. Likewise, no induction of the RecA protein in T. ferrooxidans was detected and it would be interesting to establish whether the expression of the recA gene in T. ferrooxidans is regulated by a LexA-like protein. The cloned gene was, however, expressed from either the pEcoR25 1 il pR or the pUC19 lac vector promoters. Despite changes in several amino acids that have previously been found to affect E. coli RecA protease activity, the T. ferrooxidans RecA protein has retained DNAdamage-induced proteolytic activity toward both A and the E. coli LexA repressors. These amino acid substitutions did not affect the recombinase function of the T. ferrooxidans RecA protein in E. coli (Ramesar
et al., 1988).
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