Sphaeroides Y Thioredoxin Gene - Cloning, Nucleotide Sequence, and Expression of the Rhodobacter - Journal of Bacteriology
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JOURNAL OF BACTERIOLOGY, Mar. 1990, p. 1556-1561 Vol. 172, No. 3
0021-9193/90/031556-06$02.00/0
Copyright © 1990, American Society for Microbiology
Cloning, Nucleotide Sequence, and Expression of the Rhodobacter
sphaeroides Y Thioredoxin Gene
SABINE PILLE,l* JEAN-CLAUDE CHUAT,2 ANNICK M. BRETON,' JENNY D. CLEMENT-METRAL,1
AND FRANCIS GALIBERT2
Groupe de Conception Moleculaire, Laboratoire de Technologie Enzymatique, BP649, 60206 Compiegne Cedex,1 and
Laboratoire d'Hematologie Experimentale, Centre Hayem, H6pital Saint-Louis, 75475 Paris Cedex 10,2 France
Received 31 August 1989/Accepted 15 December 1989
Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides
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Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived
from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R.
sphaeroides thioredoxin. An Ncol site was created by directed mutagenesis at the beginning of the thioredoxin
gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair
NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid
plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that
complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on
minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter
phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The
presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.
Thioredoxin is a small, ubiquitous, heat-stable protein bacteriochlorophyll membrane structures in growing photo-
with two redox-active half-cystine residues in an exposed synthetic bacteria. The thioredoxin system in R. sphaeroides
active center with the amino acid sequence Trp-Cys-Gly- Y seems to be implicated in oxygen regulation of bacterio-
Pro-Cys. This protein functions as a general protein disulfide chlorophyll synthesis by thiol redox control of the activity of
reductase in a number of oxidation-reduction reactions in- 5-aminolevulinic acid synthetase (EC 2.3.1.37), the first
volving dithiol-disulfide exchanges. Thioredoxin has been enzyme that occurs in bacteriochlorophyll synthesis. R.
isolated from many sources and has been found to partici- sphaeroides Y thioredoxin has been shown to activate
pate in a variety of biological reactions in procaryotic and purified preparations of this enzyme (4). As a first step
eucaryotic cells. The oxidized form is generally reduced by toward the understanding of an important aspect of this
NADPH and thioredoxin reductase. Reduced thioredoxin regulation, the amino acid sequence of R. sphaeroides Y
was originally isolated from Escherichia coli as an in vitro thioredoxin and a three-dimensional model derived from the
hydrogen donor for ribonucleotide reductase (18). Enzymes E. coli crystallographic structure have been previously re-
that reduce sulfate and methionine sulfoxide also use thio- ported (6).
redoxin as a hydrogen donor. Furthermore, thioredoxin was In this study, we used synthetic oligodeoxynucleotide
found to be implicated in regulation of enzyme activity by probes based on the known amino acid sequence to identify
thiol redox control (see references 11 and 13 for reviews). and clone the R. sphaeroides Y thioredoxin structural gene
Recently, an important immunological role for thioredoxin in trxA from a genomic library of this bacterium. The cloned
human lymphocytes has been suspected in view of the gene was sequenced and found to have an open reading
purification of human thioredoxin from an Epstein-Barr frame that would encode a protein identical to R. sphaeroi-
virus B-cell line and the cloning and sequencing of the des Y thioredoxin. An NcoI-PstI restriction fragment bear-
corresponding cDNA (35). Thioredoxin is also required for ing the trxA gene was ligated in an expression vector so as to
bacteriophage T7 growth and has been shown to be an be under direct control of a trc promoter. The recombinant
essential subunit of phage DNA polymerase (24). Similarly, plasmid was then introduced into a thioredoxin-deficient E.
the assembly of the filamentous phages fl and M13 requires coli mutant to produce larger quantities of R. sphaeroides
the Fip protein, which is identical to thioredoxin (22, 27). thioredoxin to further investigate structure-function relation-
Thioredoxin has been isolated from many procaryotic and ships.
eucaryotic photosynthetic organisms. The complete thiore-
doxin system (containing thioredoxin associated with MATERIALS AND METHODS
NADPH-thioredoxin reductase) in photosynthetic procary- Materials. All restriction endonucleases, calf intestinal
otes was first purified and characterized in the facultative phosphatase, and RNase A were purchased from Boehringer
phototroph Rhodobacter sphaeroides Y (5). GmbH, Mannheim, Federal Republic of Germany. T4 DNA
R. sphaeroides is a nonsulfur purple bacterium which can
grow anaerobically in light and aerobically in both light and ligase, T4 DNA polymerase, and polynucleotide kinase were
dark. Hence, R. sphaeroides provides a model system for obtained from New England BioLabs, Beverly, Mass. Ly-
the study of photosynthesis and membrane development. sozyme, isopropyl-p-D-thiogalactopyranoside, and 5-bromo-
Light intensity and molecular oxygen are the most promi- 4-chloro-3-indolyl-p-D-galactopyranoside were from Sigma
nent environmental factors that influence the synthesis of Chemical Co., Saint Louis, Mo. Bio-Gel P6 was from
Bio-Rad Laboratories, Richmond, Calif. VS-type microdial-
ysis filters were obtained from Millipore S.A., Molsheim,
*
Corresponding author. France. Colony/Plaque screen hybridization transfer mem-
1556VOL. 172, 1990 R. SPHAEROIDES Y THIOREDOXIN GENE 1557
branes, [-y-32P]ATP, and [a-35S]dATP were purchased from Oligonucleotides. Oligodeoxynucleotides were synthesized
Dupont, NEN Research Products, Boston, Mass. Hybond- by the phosphoramidite method with an 8600 Biosearch
N hybridization transfer membranes were obtained from automated DNA synthesizer. After deprotection, they were
Amersham International, Amersham, Buckinghamshire, En- purified by acrylamide gel electrophoresis. Oligonucleotide
gland. probe labeling was performed with [y-32P]ATP by using T4
Bacterial strains, phages, and plasmids. E. coli MV1190 polynucleotide kinase as described by Maniatis et al. (23).
[A(lac-proAB) thi supE A(srl-recA)306: :TnJO (Tetr)(F' We obtained a specific activity of about 108 cpm/,g of DNA.
traD36 proAB lacIq ZAM15)] was used for transfection with 32P-labeled probes were separated from free [y-32P]ATP by
bacteriophage M13 and transformation. E. coli LE392 Bio-Rad P6 column chromatography.
[hsdR514 supE44 supF58 lacYJ or A(lacIZY) galK2 galT22 Purification of plasmid DNA. Plasmid DNA was isolated
metBI trpR55] and P2392, which is LE392 with phage P2, and purified from fresh overnight cultures by the boiling
were used as hosts for bacteriophage lambda and were method described by Maniatis et al. (23), except that phenol-
obtained from Stratagene, La Jolla, Calif. E. coli CJ236 chloroform (1:1) extraction was performed once before
[dut-J ung-J thi-J relAJ(pCJ105) (Cmr)] was used for direct- ethanol precipitation in 0.3 M ammonium acetate.
Subcloning in bacteriophage M13 and sequencing. Positive
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ed-mutagenesis experiments with recombinant phage M13 as
described by the manufacturer (Bio-Rad). E. coli K-12 strain phage clones harvested from cultures of E. coli P2392 were
BH216 (F- araD139? galU galK hsr strA metE46 argH1 purified as described by Ausubel et al. (3). Lambda DNA
ilvC::TnS trxA2) was used to detect expression of recombi- was extracted, and the insert was excised by BamHI. The
nant plasmid pKK233-2::trxA (named pUTC3) and was a resulting fragments were ligated to dephosphorylated
generous gift from J. A. Fuchs. Strain BH216F', which is M13mp18 replicative-form DNA cut by BamHI and were
strain BH216 with an F' factor (proAB lacIq ZAM15 TnJO) used to transfect MV1190 cells after preparation of compe-
extracted from strain XLI-BLUE was used for M13 infection tent cells by the method of Hanahan (12). Single-stranded
tests. E. coli SH250 (ilvD145 metE46 trp pro mtl malA ara DNA from recombinant phage M13 was isolated as de-
gal lac recAl rpsLl tonA btuB tsx TnlO?) was also used. E. scribed by Sanger et al. (29). Sequencing was performed by
coli strains were grown in liquid culture in LB medium (23) the dideoxy-chain termination method by using the Seque-
supplemented with 10 mM MgCl2 and 0.2% maltose for nase kit from United States Biochemical Corp., Cleveland,
strains LE392 and P2392. When needed, ampicillin was Ohio, with a synthetic 17-base universal primer or specific
added to the medium to a final concentration of 100 ,ug/ml. primers.
Minimal medium was M9 medium (23) supplemented with 50 In vitro directed mutagenesis. A mutagenic, synthetic
pRg of the amino acid required by auxotrophic strains per ml. 26-mer oligodeoxynucleotide (SPNCO) was annealed to the
Phage lambda EMBL3 (Stratagene) was used for genomic single strand of the recombinant template M13mpl8 bearing
library construction. Phage M13mpl8 was used for sequenc- the R. sphaeroides thioredoxin gene. The Muta-Gene kit
ing, and plasmid pKK233-2 was used as an expression vector from Bio-Rad, based on a method described by Kunkel (17),
for the cloned thioredoxin gene of R. sphaeroides in E. coli. was used as specified by the manufacturer for annealing and
M13mp18 and pKK233-2 were obtained from Pharmacia, extending conditions and for selection of the mutagenized
Uppsala, Sweden. Recombinant DNA constructions involv- strand.
ing phage M13 and its derivatives were screened with 40 RI Thioredoxin assay. Activity of thioredoxin was assayed as
of 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside at a described by Cldment-Metral et al. (7), except that purified
concentration of 4% in dimethylformamide in 40 RIl of 0.1 M thioredoxin was replaced by crude cell extracts. This
isopropyl-,-D-thiogalactopyranoside mixed with 3.5 ml of method is based on the reduction of 5,5'-dithiobis(2-ni-
0.8% agarose for detection of insertional inactivation of trobenzoic acid) by reduced thioredoxin produced by
P-galactosidase (25). Phage T7, obtained from A. Holmgren, NADPH and thioredoxin reductase. The reaction was mon-
was used for infection tests. itored at 412 nm in a Cary 2300 spectrophotometer. Prepa-
Genomic library construction in lambda EMBL3. R. ration of the crude cell extract included passage through a
sphaeroides Y (wild type) was grown in rich YCC medium at French press to break the cells. Crude cell extracts were
37°C with aeration (31) and harvested during the late-expo- heated for 10 min at 80°C to inactivate interfering enzymes
nential growth phase. Total DNA from R. sphaeroides was and then cooled on ice. The suspension was centrifuged for
extracted by sodium dodecyl sulfate-phenol as described by 40 min at 19,000 rpm in a Beckman JA20 rotor, and the
Davis et al. (8) and partially digested with endonuclease supernatant was stored at -20°C.
Sau3A at 2.8 x 10-3 U/mg of DNA. Fragments were size
separated on a 5 to 30% sucrose-1 M NaCl gradient by RESULTS
centrifugation in an SW41 Beckman rotor at 18,000 rpm for
20 h. The 9- to 20-kb Sau3A DNA fragments of R. sphaeroi- Isolation of recombinant phage lambda that hybridized with
des were microdialyzed on Millipore type VS membranes for the oligonucleotide probes. Two oligodeoxynucleotides de-
2 h at room temperature (30). Ligatures of R. sphaeroides signed from the known amino acid sequence of R. sphaeroi-
DNA onto EMBL3 BamHI arms and packaging with an in des thioredoxin were synthesized in the same orientation as
vitro Gigapack gold packaging kit from Stratagene were the noncoding strand of the structural gene. The first oligo-
performed as recommended by the manufacturer. The nucleotide, designated SP1, corresponded to the sequence
recombinant phages were selected by plating on P2392 strain between amino acids 24 and 30, and the second (SP2)
and plaque purified. corresponded to the sequence between amino acids 55 and
Hybridization. DNA transfers from agarose gels onto 62; both were 23 nucleotides long (Fig. 1A). After ligation
nylon filters were performed as described by Southern and and packaging of 9- to 20-kb Sau3A-cut chromosomal DNA
adapted by Amersham, with Hybond nylon filters. Replicas fragments in lambda phage EMBL3 BamHI arms, 20,000
of bacteriophage plaques were made on nylon membranes. recombinant phage were obtained on strain P2392. This
All hybridizations with 3 ng of 32P-end-labeled probes per ml result is comparable to that obtained with A L47-1 by Muller
were performed at 40°C as recommended by Amersham. et al. (26). The genomic library was screened by primary1558 PILLE ET AL. J. BACTERIOL.
ably indicates incomplete EcoRI digestion. Digestion of X Dl
DNA by BamHI generated six fragments, among which one
3.5 kilobases (kb) long hybridized to oligonucleotide SP1.
Southern analysis of a BamHI hydrolysate of total genomic
DNA also revealed a positive signal at 3.5 kb, suggesting that
the trxA gene was carried by the 3.5-kb fragment (data not
shown). In light of these results and since PstI and HindIII
digestions produced DNA fragments that were too large, the
3.5-kb BamHI fragment that hybridized with the SP1 probe
was selected for further subcloning into M13mpl8.
Subcloning into M13mpl8 for sequencing and directed-
mutagenesis experiments. BamHI-cut X Dl fragments were
ligated into dephosphorylated BamHI-cut M13mpl8 replica-
tive-form DNA and used to transfect strain MV1190 bacte-
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ria. M13 recombinants into which the 3.5-kb BamHI-cut X
Dl fragment had been inserted were screened by plaque
hybridization with both oligonucleotide probes. The positive
plaques were picked up. Templates were prepared and
FIG. 1. (A) Sequences of the synthetic oligonucleotides used for checked by dot blot hybridization with oligonucleotide SP1.
genomic library screening. Oligonucleotide sequences were de- Sequencing was performed with Sequenase as described by
tected on the basis of the known amino acid sequence. (B) Identi- United States Biochemical with [35`]dATP using various
fication of the R. sphaeroides trxA gene inserted in recombinant oligonucleotides as specific primers to sequence the part of
clone X Dl by use of specific oligonucleotide probe SP1 in Southern the 3.5-kb insert where the trxA gene is (Fig. 2). Primer
hybridization blots of restriction fragments of X Dl. The same extension of oligonucleotide SP1 provided the sequence of
amount of X Dl DNA was treated with restriction endonuclease the 5' region of the gene, from which another oligonucleotide
EcoRI (lane 1), BamHI (lane 2), PstI (lane 3), or HindIII (lane 4). (SP3) was synthesized to isolate the complementary tem-
Sizes are indicated in kilobases. An identical pattern was obtained
when the same Southern blot was hybridized to oligonucleotide plate designated M13UTC3-2 (the first template was previ-
probe SP2; however, the autoradiographic signal was slighter for ously designated M13UTC3-1) and sequence the opposite
every band. strand. As sequencing progressed, new oligonucleotides and
the universal 17-mer primers were used to obtain the com-
plete gene sequence. The nucleotide sequence determined in
plaque hybridization with oligonucleotide probes SP1 and this way from both strands and the corresponding deduced
SP2. One plaque displayed a positive signal with both amino acid sequence are shown in Fig. 3. The genomic
probes. The corresponding recombinant phage, X Dl, was sequence confirms the amino acid sequence previously re-
picked, eluted, and plated for purification by secondary ported (6) and removes the ambiguity about amino acid 63,
plaque hybridization. After amplification, the phage was which is glutamic acid and not glutamine.
harvested by elution. X Dl recombinant clone DNA was A possible Shine-Dalgarno ribosome-binding site (AGG
purified on a CsCl gradient, extracted, and digested by AG) is 8 nucleotides distant from the initiation codon. The
restriction endonucleases EcoRI, HindIII, PstI, and BamHI. sequence downstream of the coding region contains a stable
The Southern hybridization blot pattern of A Dl DNA with stem-and-loop structure (AG, -18.4 kcal [1 cal = 4.184 J])
oligonucleotide probe SP1 is shown in Fig. 1B. Two EcoRI with seven G-C pairs, beginning at position 405, followed by
fragments hybridized with the SP1 probe, which most prob- a T-rich region. This stem-and-loop structure resembles a
0.25Kb
BamHl BamHdI
I
0ff
MI3UTC3.1
7-7-7-1 1I !K 1 -
, 40bp
0 1464bp
ATG~
AM TGA
5 L- 1
SP3 SP7
S4-
spi SP2 UP
FIG. 2. Positions on the sequenced part of the M13UTC3-1 clone of synthetic oligonucleotides. Oligonucleotides SP1 and SP2 were used
for genomic library screening. SP3 and SP7 were used as primers for trxA gene sequencing. The universal 17-mer primer (UP) was also used
to sequence in the opposite direction. SP1 was used for both screening and sequencing. The hatched line represents M13mpl8 DNA, and the
thin line is R. sphaeroides DNA. The double line represents the sequenced fragment; the ATG initiation codon and the TGA termination
codon are indicated. The arrows indicate the direction and extent of sequencing.VOL. 172, 1990 R. SPHAEROIDES Y THIOREDOXIN GENE 1559
10
GOCMGO.cGGACCw.AT 'ATG TCC ACC GT CCC GTG ACG GAC
so Nco I Pst I
M S T V P V T D 42tbp
100 I. i
GCC ACC TTC GAC C GM C0C AAG TCC GAC G CCC GT GS GM GM ST GG GC GAA GG S- 9
A T F D T E V R K S D V P V V V D F W A E W frxA
200
TOR ,'
1S0
TGC GGC TGC CGG CAG ATC GGC CC GCG CTC GAG GG CTC TCG AM GMA GCC GGC AAG G
C G
CCC
P C R Q I G P A L E E L S K E Y A G K V 4Obp
a.-
250
AAG TC GTG
A AM G AAT GM GCM GA TAC CCC GG MC CCG GCC ATO CTG GGC rCCC GM ATC
K I V K V N V D E N P Z S P A M L G V R G I
300
COG GCG CSG GTC CTG GMC AAG AAC GGT CMG GTC GTG TCG AAC AAG GTC GGC OCT GCG CCG AAG GMC e
P A L F L F K N G Q V V S N K V G A A P K A
350 400
GCG CTG GMC XX TWG ATC OCT G GCC CTC TGA
A L A T I A S A L
450
FIG. 3. Nucleotide and encoded amino acid sequences of the
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thioredoxin gene from R. sphaeroides Y. The ribosome-binding site
upstream of the initiator ATG is underlined. The region of inverted
repeats is depicted by opposing arrows. The asterisk indicates the
termination codon.
rho-independent transcription termination signal. A possible
second stem-and-loop structure (AG, -10.4 Kcal) is located
upstream at position 382.
The R. sphaeroides thioredoxin gene displays highly se-
lective codon usage. For example, of the six leucine codons,
only two are used; of the four glycine codons, the GGC FIG. 4. Construction of vector pUTC3. Mutagenized double-
codon is preferentially used; and for isoleucine the ATC stranded M13UTC3-1N DNA was hydrolyzed by endonucleases
codon is used exclusively. The third position is occupied by NcoI and PstI, giving a 421-bp insert containing the R. sphaeroides
G or C in more than 90% of the total codons. Overall, the trxA gene with the 86-bp downstream sequence, followed by 17 bp
G+C content is 66%. This result is comparable to the base of M13mp18 DNA. This 421-bp insert was then ligated into the
NcoI-PstI-cut pKK233-2 vector. The ligated mixture was used to
composition of R. sphaeroides chromosomal DNA, which transform strain MV1190 to select recombinant plasmids by ampi-
displays 69% G+C (33). cillin resistance.
Cloning in an expression vector. pKK233-2 is an E. coli
gene expression plasmid with a trc promoter and an ATG
translation initiation codon within a unique NcoI restriction R. sphaeroides NADPH-thioredoxin reductase (Table 1). In
site 8 bases downstream of an AGGA Shine-Dalgarno se- the reaction involving crude extract from the BH216
quence. The presence of an NcoI site at this position allows (pUTC3) transformant clone, the specific activity of thiore-
for direct insertion of a gene into this vector at an optimal doxin was higher when R. sphaeroides rather than E. coli
distance from the trc promoter and in the correct reading thioredoxin reductase was used, thus indicating the presence
frame (1). To insert the trxA gene directly in phase with the of R. sphaeroides thioredoxin encoded by plasmid pUTC3
vector promoter, an NcoI restriction site had to be created bearing the R. sphaeroides trxA gene. The activity of this
by directed mutagenesis. Three nucleotides had to be protein did not seem to be altered by replacement of Ser in
changed at positions 55, 56, and 60 (Fig. 3) to obtain an NcoI position 2 by Ala, as the ratio of the activities assayed with
site at the ATG initiation codon. The mutation at position 60 the two different thioredoxin reductases was in good agree-
induced the replacement of Ser in position 2 by Ala in the
encoded protein. The mutagenic oligonucleotide SPNCO,
containing the desired mismatch relative to the cloned DNA, TABLE 1. Enzymatic assay of thioredoxin
was annealed to the uracil-containing DNA M13UTC3-1
template. Of 12 plaques tested by DNA sequencing, 4 were Sp act with thioredoxin
Thioredoxin concn
Source of crude reductase fromb:(p/gotta
found to be mutated; no other mutation was observed. After extracta (,ug/mg of total
purification, a 421-base-pair (bp) restriction fragment of the E. coli R. sphaeroides protein)
mutagenized double-stranded M13UTC3-1N DNA was li- E. coli
gated into vector pKK233-2 (Fig. 4). Eight plasmids ex- SH250 (trxA+) 11.7 1.6 4.4
tracted from Ampr transformants of strain MV1190 gave a BH216 (trxA) 2.2 0.4 NRc
positive signal when hybridized with 32P-labeled oligonucle- BH216(pUTC3) 32.2 56.5 24.3
otide probe SP7. Restriction enzyme analysis of one of the
plasmids (pUTC3) showed that it was inserted in the correct R. sphaeroides 2.3 3.6 1.5
orientation with regard to the trc promoter. This hybrid a
The assay was performed with 100 ,ul of extract at the following protein
plasmid was used to transform E. coli trxA2 mutant strains concentrations (A280 units): SH250, 22.8; BH216, 22.8; BH216(pUTC3), 35;
BH216 and BH216F'. R. sphaeroides, 56.
R. sphaeroides thioredoxin assay and biological tests with E. b Specific activities are expressed as 102 U/mg of total protein in the extract.
coli. The assay of thioredoxin from crude cell extracts used One unit of activity corresponds to a AA412 per minute of 1; A280 = 1 is taken
as equivalent to 1 mg of protein per ml. The reaction mixture contained 10 F.M
the cross-reactivity of R. sphaeroides thioredoxin with E. thioredoxin reductase from either E. coli or R. sphaeroides, corresponding,
coli NADPH-thioredoxin reductase (7), and similarly, thio- respectively, to 185 and 53 U/mg as measured with pure E. coli thioredoxin.
redoxin from E. coli SH250 (trxA+) was assayed with pure c NR, Not relevant.1560 PILLE ET AL. J. BACTERIOL.
ment with the ratio expected from the kinetic constants in place of the R. sphaeroides enzyme. This latter result is
measured in cross-reactivity tests with the pure proteins (7). comparable to that obtained with pure thioredoxin (7). Thus,
The nonspecific background reaction was represented by a detection of thioredoxin activity with both thioredoxin re-
reaction involving thioredoxin-deficient E. coli BH216 with ductases is a good assay to evaluate the amount of expressed
either E. coli or R. sphaeroides thioredoxin reductase. R. sphaeroides protein in a transformant clone (Table 1).
To determine whether R. sphaeroides thioredoxin can Since plasmid extraction of expression vector pKK233-2,
function in vivo in E. coli cells, we tested the complemen- which bears the R. sphaeroides trxA gene inserted in reverse
tation of several phenotypes in thioredoxin-deficient mu- orientation produced a higher amount of plasmid DNA than
tants. BH216(pUTC3) clones exhibited a larger colony size extraction of the vector with the trxA gene in the correct
than the trxA-carrying parent on LB medium and, further- orientation with the trc promoter (data not shown), it can be
more, grew as well as wild-type strain MV1190 with 30 ,ug of suggested that strains or plasmids in which R. sphaeroides
methionine sulfoxide per ml as the methionine source on trxA is expressed are unstable. Attempts will be made to
minimal M9 medium, whereas trxA metE46-carrying parent stabilize strains containing the R. sphaeroides trxA gene on
strain BH216 was unable to use methionine sulfoxide (28). an expression vector. Derivatives of a trxA-carrying E. coli
The latter result showed that R. sphaeroides thioredoxin strain containing the R. sphaeroides Y gene appear to carry
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functions as a cofactor for E. coli methionine sulfoxide trxA+ for all of the phenotypes observed. Although in the
reductase in vivo. Unlike BH216, transformed strain cloned R. sphaeroides Y thioredoxin the Ser in position 2 is
BH216(pUTC3) was able to support T7 phage replication as replaced by Ala, the activity of the protein appears to be
well as was wild-type strain MV1190, indicating that R. unchanged. Unlike Anabaena sp. strain PCC7119 and Cory-
sphaeroides thioredoxin is able to serve as a subunit for the nebacterium nephridii C-2 thioredoxins expressed from plas-
gene 5 protein to produce active T7 DNA polymerase. mids introduced into a trxA-carrying E. coli strain, R.
Similarly, filamentous phage M13mpl8 was able to grow on sphaeroides thioredoxin can support the growth of phage T7
BH216F'(pUTC3), although the plaques formed were more in strain BH216(pUTC3). Thioredoxin is an essential subunit
turbid than on a strain with E. coli thioredoxin. Thus, R. of phage T7 DNA polymerase (24). By binding to gene 5
sphaeroides thioredoxin can replace E. coli thioredoxin in protein of phage T7, thioredoxin acts as an accessory protein
the phage assembly process. and confers processibility on the polymerizing reaction (32).
Eklund et al. (10) have identified a "molecular surface area"
DISCUSSION that, as they suggest, forms the binding surface for interac-
tions between thioredoxin and other proteins. This area is
We have identified the R. sphaeroides Y thioredoxin gene composed of three regions around residues 33 and 34, 74 to
by using synthetic oligodeoxynucleotides based on the 76, and 91 to 93. Mutants have been described which failed
known acid sequence as hybridization probes from a ge- to support filamentous phage assembly and T7 growth (14,
nomic library of R. sphaeroides Y. Successful cloning was 28). The location of these mutations at or adjacent to the
confirmed by both DNA sequence analysis and in vivo residues identified by Eklund et al. (10) suggests that this
expression of thioredoxin in E. coli. Synthetic oligodeoxy- surface area is critical for thioredoxin interaction with the
nucleotides have also been used to isolate some R. sphaeroi- gene 5 protein (14). Comparison of the amino acid sequence
des gene, such as the R. sphaeroides 2.4.1 cytochrome c2 of R. sphaeroides thioredoxin with other known thioredoxin
gene (9) and the genes that encode the B800-850 light- sequences (19-21, 34) shows that, at the position corre-
harvesting complex of R. sphaeroides (2, 16). Codons used sponding to residue 74 of E. coli thioredoxin, Anabaena
in the R. sphaeroides trxA gene compare well to those in PCC7119 and C. nephridii C-2 have Ser and Asn, respec-
other R. sphaeroides genes that have been previously se- tively, whereas both E. coli and R. sphaeroides have a Gly
quenced (2, 9, 15, 16). Generally, there is preferentially a G residue. Moreover, residues 73 to 76 are identical in the R.
or a C in position 3 (and in other positions for amino acids sphaeroides and E. coli thioredoxins. Since both thioredox-
with six codons), in agreement with the G+C content of the ins can also support T7 replication, it can be proposed that
R. sphaeroides genome. The proportion of infrequently used this surface region, particularly residue Gly-74, is important
codons based on the codon usage of E. coli is 32% for the R. in the interaction between the gene 5 protein and thiore-
sphaeroides trxA gene, whereas it is 11% for the E. coli gene. doxin.
It can be suggested that the choice of optimal or nonoptimal For understanding of the role of thioredoxin in the oxygen
codons in R. sphaeroides may be different from that in E. regulation of bacteriochlorophyll synthesis, gene cloning will
coli. Nevertheless, the coding sequences of E. coli and R. allow, through mutagenesis, in vivo investigations of this
sphaeroides trxA genes display 60% homology, leading to protein in the photosynthetic bacterium R. sphaeroides.
49% amino acid sequence identity (of 105 residues) when the
alignment is centered on the active-site disulfide (6). This ACKNOWLEDGMENTS
result, with additional amino acid comparison with other
thioredoxins, lends support to the suggestion of Lim et al. We gratefully thank Luc d'Auriol for the synthesis of oligonucle-
(21) that thioredoxins have evolved from the same ancestral otides and Catherine Pilon for skillful assistance with DNA sequenc-
gene. ing. We acknowledge Fr6d6ric Ampe for help with the expression
tests. We also thank Simon Gamble and Sally Taylor for excellent
A thioredoxin assay based on the cross-reactivity of R. work on the thioredoxin assay.
sphaeroides thioredoxin with E. coli thioredoxin reductase This work was supported by the Ministere de la Recherche et de
confirmed the expression of recombinant plasmid pUTC3. l'Enseignement Scientifique and the Centre National de la Recher-
Crude extract from a BH216(pUTC3) transformant clone che Scientifique (UPR 41, URA 523).
showed a twofold increase of specific activity when the
reaction involved R. sphaeroides thioredoxin reductase in LITERATURE CITED
place of the E. coli enzyme. In contrast, crude extract from 1. Amann, E., and J. Brosius. 1985. ATG vectors for regulated
E. coli SH250 (trxA+) showed a sevenfold increase of high-level expression of cloned genes in Escherichia coli. Gene
specific activity when E. coli thioredoxin reductase was used 40:183-190.VOL. 172, 1990 R. SPHAEROIDES Y THIOREDOXIN GENE 1561
2. Ashby, M. K., S. A. Coomber, and C. N. Hunter. 1987. Cloning, tion of thioredoxin, the hydrogen donor of Escherichia coli B. J.
nucleotide sequence and transfer of genes for the B800-850 Biol. Chem. 239:3436-3444.
light-harvesting complex of Rhodobacter sphaeroides. FEBS 19. Lim, C.-J., J. A. Fuchs, S. C. Mc Farlan, and H. P. C. Hogen-
Lett. 213:245-248. kamp. 1987. Cloning, expression and nucleotide sequence of a
3. Ausubel, F. M., R. R. Brent, R. E. Kingston, D. D. Moore, J. A. gene encoding a second thioredoxin from Corynebacterium
Smith, J. C. Fiedman, and K. Struhl. 1987. Current protocols in nephridii. J. Biol. Chem. 262:12114-12119.
molecular biology. John Wiley & Sons, Inc., New York. 20. Lim, C.-J., D. Geraghty, and J. A. Fuchs. 1985. Cloning and
4. Clement-Metral, J. D. 1979. Activation of ALA synthetase by nucleotide sequence of the trxA gene of Escherichia coli K-12.
reduced thioredoxin in Rhodopseudomonas sphaeroides Y. J. Bacteriol. 163:311-316.
FEBS Lett. 101:116-120. 21. Lim, C.-J., F. K. Gleason, and J. A. Fuchs. 1986. Cloning,
5. Clement-Metral, J. D., and A. Holmgren. 1983. Purification and expression, and characterization of the Anabaena thioredoxin
some properties of thioredoxin from a photosynthetic bacte- gene in Escherichia coli. J. Bacteriol. 168:1258-1264.
rium: Rhodopseudomonas sphaeroides Y. Colloq. Int. Cent. 22. Lim, C.-J., B. Haller, and J. A. Fuchs. 1985. Thioredoxin is the
Natl. Rech. Sci., p. 59-68. bacterial protein encoded by fip that is required for filamentous
6. Clement-Metral, J. D., A. Holmgren, C. Cambillau, H. Jornvall, bacteriophage fl assembly. J. Bacteriol. 161:799-802.
H. Eklund, D. Thomas, and F. Lederer. 1988. Amino acid 23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
Downloaded from http://jb.asm.org/ on February 10, 2021 by guest
sequence determination and three-dimensional modelling of cloning: a laboratory manual. Cold Spring Harbor Laboratory,
thioredoxin from the photosynthetic bacterium Rhodobacter Cold Spring Harbor, N.Y.
sphaeroides Y. Eur. J. Biochem. 172:413-419. 24. Mark, D., and C. C. Richardson. 1976. Escherichia coli thiore-
7. Clement-Metral, J. D., J. 0. Hoog, and A. Holmgren. 1986. doxin: a subunit of bacteriophage T7 DNA polymerase. Proc.
Characterization of the thioredoxin system in the facultative Natl. Acad. Sci. USA 73:780-784.
phototroph Rhodobacter sphaeroides Y. Eur. J. Biochem. 161: 25. Messing, J. 1983. New M13 vectors for cloning. Methods
119-126. Enzymol. 101:20-79.
8. Davis, R. W., D. Botstein, and J. R. Roth. 1980. A manual for 26. Muller, E. D., J. Chory, and S. Kaplan. 1985. Cloning and
genetic engineering. Advanced bacterial genetics, p. 120-123. characterization of the gene product of the form II ribulose-
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1,5-bisphosphate carboxylase gene of Rhodopseudomonas
9. Donohue, T. J., A. G. McEwan, and S. Kaplan. 1986. Cloning, sphaeroides. J. Bacteriol. 161:469-472.
DNA sequence, and expression of the Rhodobacter sphaeroides 27. Russel, M., and P. Model. 1985. Thioredoxin is required for
cytochrome c2 gene. J. Bacteriol. 168:962-972. filamentous phage assembly. Proc. Natl. Acad. Sci. USA 82:
10. Eklund, H., C. Cambillau, B. M. Sjoberg, A. Holmgren, H. 29-33.
Jornvall, J. 0. Hoog, and C. I. Branden. 1984. Conformational 28. Russel, M., and P. Model. 1986. The role of thioredoxin in
and functional similarities between glutaredoxin and thioredox- filamentous phage assembly. J. Biol. Chem. 261:14997-15005.
ins. EMBO J. 3:1443-1449. 29. Sanger, F., A. R. Coulson, B. G. Barrell, A. J. H. Smith, and
11. Gleason, F. K., and A. Holmgren. 1988. Thioredoxin and related B. A. Roe. 1980. Cloning in single-stranded bacteriophage as an
aid to rapid DNA sequencing. J. Mol. Biol. 143:161-178.
proteins in procaryotes. FEMS Microbiol. 54:271-298. 30. Silhavy, T. J., M. N. Berman, and L. W. Enquist. 1984.
12. Hanahan, D. 1983. Studies on transformation of Escherichia coli Experiments with gene fusions, p. 182. Cold Spring Harbor
with plasmids. J. Mol. Biol. 166:557-580. Laboratory, Cold Spring Harbor, N.Y.
13. Holmgren. A. 1985. Thioredoxin. Annu. Rev. Biochem. 54: 31. Sistrom, W. R. 1977. Transfer of chromosomal genes mediated
237-271. by plasmid R6845 in Rhodopseudomonas sphaeroides. J. Bac-
14. Huber, H. E., M. Russel, P. Model, and C. C. Richardson. 1986. teriol. 131:526-532.
Interaction of mutant thioredoxins of Escherichia coli with the 32. Tabor, S., H. E. Hubert, and C. C. Richardson. 1987. Esche-
gene S protein of phage T7. J. Biol. Chem. 261:15006-15012. richia coli thioredoxin confers processivity on the DNA poly-
15. Kiley, P. J., T. J. Donohue, W. A. Havelka, and S. Kaplan. 1987. merase activity of the gene 5 protein of bacteriophage T7. J.
DNA sequence and in vitro expression of the B875 light- Biol. Chem. 262:16212-16223.
harvesting polypeptides of Rhodobacter sphaeroides. J. Bacte- 33. Truper, H. G., and N. Pfenig. 1978. Taxonomy of the Rhodos-
riol. 169:742-750. pirillales, p. 19-27. In R. K. Clayton and W. R. Sistrom (ed.),
16. Kiley, P. J., and S. Kaplan. 1987. Cloning, DNA sequence, and The photosynthetic bacteria. Plenum Publishing Corp., New
expression of the Rhodobacter sphaeroides light-harvesting York.
complex B800-850-a and B800-850-P genes. J. Bacteriol. 169: 34. Wallace, B. J., and S. R. Kushner. 1984. Genetic and physical
3268-3275. analysis of the thioredoxin (trxA) gene of Escherichia coli K-12.
17. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis Gene 32:399-408.
without phenotypic selection. Proc. Natl. Acad. Sci. USA 35. Wollman, E. E., L. d'Auriol, L. Rimsky, A. Shaw, J. P. Jacquot,
82:488-492. P. Wingfield, P. Graber, F. Dessarps, P. Robin, F. Galibert, J.
18. Laurent, T. C., E. C. Moore, and P. Reichard. 1964. Enzymatic Bertogliot, and D. Fradelizi. 1988. Cloning and expression of a
synthesis of deoxyribonucleotides. Isolation and characteriza- cDNA for human thioredoxin. J. Biol. Chem. 263:15506-15512.You can also read
