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(Received for publication, September 21, 1995) From the
We report the identification of the gene encoding a DNA
photolyase (phrA) from the Gram-negative eubacterium Myxococcus xanthus. The deduced amino acid sequence of M.
xanthus photolyase indicates that the protein contains 401 amino
acids (M
Photolyases play an important role in the repair of damage to
DNA by ultraviolet radiation(1) . The sequences of photolyase
genes from numerous organisms, both prokaryotic and eukaryotic, have
been reported (for summaries, see (2) and (3) ).
Although all known photolyases share some similarities in amino acid
sequence, they appear to fall into two distinct classes in which the
photolyases within a class show strong amino acid sequence similarities
but show weak similarity to members of the other class. Photolyases
from a number of diverse microorganisms including both fungi and
eubacteria constitute one class. The other class includes photolyases
from a teleost fish, a marsupial, an insect, and an Archaeum (Methanobacterium thermoautotrophicum)(3) . In this
paper, we describe the identification and characterization of phrA from the Gram-negative eubacterium Myxococcus xanthus. M. xanthus is a soil-dwelling eubacterium that has
light-inducible carotenoid pigments for protection from photolysis (4) . Cells secrete catabolic enzymes to extracellularly digest
their prey and macromolecules in their environment. If a population of
cells becomes starved for any one of several essential nutrients,
aggregates of 10
Figure 1:
Cloning of M. xanthus phrA.
Standard recombinant DNA techniques were used to subclone a BamHI-PstI M. xanthus DZF1 fragment from pBB12 (10) into pUC118 and pUC119 for single-stranded DNA
sequencing(9) . Note that the lacZ and phrA promoters are both in a ``clockwise'' orientation in
pBB12 and pML118. The two promoters are in opposite orientations in
pMW119.
Figure 2:
Sequence of M. xanthus phrA. Both
strands of the 2.2-kilobase pair BamHI-PstI fragment
were fully sequenced as described under ``Materials and
Methods.'' The figure shows 1.92 kilobase pairs of DNA proximal to
the BamHI site, including the photolyase open reading frame
and upstream regulatory sequences. Amino acids are designated by the single-letter code. The residue identified by primer extension
as the start of transcription for phrA is underlined and italicized at nucleotide 486. The deduced promoter
sequences are boxed, beginning at residues 453(-35) and
476(-10). The presumptive ribosome binding site is shown in underlined, bold letters beginning at residue 613.
The assumed beginning of translation is shown by an overhead arrow at residue 623 for
PhrA.
Fig. 3shows the alignment of M. xanthus to six other photolyases to which BLAST found similarity (Table 4): the four photolyases to which it is most similar, as
well as the photolyase of Streptomyces griseus (the
eubacterial photolyase to which BLAST found it to be most similar) and
the photolyase of E. coli for which the crystal structure has
recently been published(37) . As predicted by BLAST, M.
xanthus photolyase is more similar to the four photolyases of
``higher'' eukaryotic origin than to the two eubacterial
photolyases. Note that the region of similarity at the amino terminus
extends into the beginning of the open reading frame of M. xanthus which is unlikely to be translated. No DNA homology was detected
in this region (MACAW, data not shown). Based upon the alignment shown
in Fig. 3, M. xanthus photolyase was found to be
15-16% identical to the eubacterial photolyases while it is 30%
identical to the eukaryotic photolyases shown (Table 6). As shown
in Fig. 3, the carboxyl terminus is the most highly conserved
region of photolyase. An analysis of the DNA in this region using MACAW
finds that among these seven distantly related organisms there is still
a conserved region of homology among the
eukaryotic-Methanobacterium group of photolyases and the
photolyase of M. xanthus (Fig. 4). MACAW also found
significant homology between S. griseus and E. coli DNAs in the region shown in Fig. 4. However, MACAW did not
detect significant homology between M. xanthus and either of
the other two eubacterial DNAs. The homology among the group including M. xanthus persists even though the G+C content of the
other genes relative to M. xanthus is markedly different (Table 6). It has been suggested that the differences between
this group of photolyases and the eubacterial photolyases in this
region of the DNA arose through a deletion(2) .
Figure 3:
Alignment of M. xanthus photolyase to other photolyases. MACAW was used to align the open
reading frame containing M. xanthus photolyase to the
published sequences of the four photolyases most similar to the
photolyase of M. xanthus and to the most similar photolyase
from eubacteria (S. griseus) as well as the photolyase of E. coli (see Table 4). Accession numbers for the
different genes are given in the legend to Table 4. Amino acids
are denoted by the single-letter code. Identical amino acids
are denoted a white letter on a black background.
Non-identical, conservative changes (BLOSUM62 values
Figure 4:
DNA homology in the carboxyl terminus of
photolyases from diverse organisms. MACAW was used to align the DNA
sequences of the seven photolyases shown in Fig. 3. Significant
homology was detected among Monodelphis domestica, Drosophila melanogaster, Carassius auratus, M.
thermoautotrophicum, and M. xanthus. MACAW did not detect
homology between this group and either of the other two eubacterial
sequences. MACAW did detect homology between E. coli and S. griseus in this region. Sequences are aligned in accordance
with the alignment of the amino acid sequence in Fig. 3.
Accession numbers for the genes are given in the legend to Table 4.
In this paper we describe an open reading frame, ORF7, which
encodes a DNA photolyase (phrA) from M. xanthus.
Although the M. xanthus photolyase is not significantly
similar to that of E. coli, the cloned gene does,
nevertheless, rescue photoreactivation in E. coli.The
increased sensitivity of E. coli SY2 (phrA The crystal structure of photolyase from E. coli has recently been published(37) . When the
regions of similarity of the M. xanthus photolyase were
compared to the structure of E. coli photolyase, it was found
that 8 of the 13 amino acids involved in the FAD binding site are
identical. An additional 3 of the 13 are conserved (Fig. 3).
This suggests that the FAD binding site of photolyases has been
conserved throughout evolution. We do not know if M. xanthus photolyase belongs to the group of photolyases, including that
from E. coli, which use 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) as the second
chromophore. Therefore, the state of conservation of the MTHF binding
site is unclear. GCG Peptidestructure (see ``Materials and
Methods'') was used to predict the structure of M. xanthus and E. coli photolyases. It correctly predicted the
pattern of alternating The conservation of an untranslated amino
terminus of the open reading frame containing phrA was
unexpected. MACAW and visual inspection detect no significant homology
of the DNAs (data not shown). The evidence that this region is
untranslated is strong. (i) Primer extension shows the beginning of the
mRNA to be internal to this region; (ii) there is a good promoter
associated with the start of transcription, but there is no other
identifiable promoter 5` to this region; and (iii) the first potential
ribosome binding site associated with this region is associated with an
appropriate start codon (ATG) at amino acid 68 of ORF7 (Fig. 2).
What is the selective pressure for this conservation? Perhaps there has
been an evolutionarily recent rearrangement in the 5` region of the M. xanthus gene with very little drift in the region that is
now the promoter and no longer translated. The pattern of alternating
Codon usage by phrA is
consistent with that observed for other M. xanthus genes(20) . It was hoped that the CAI would enable us to
estimate if the amount of photolyase in the cell was regulated by the
use of rare codons as described for E. coli(19, 39) . However, the data base for determining
the CAI for M. xanthus open reading frames is not large enough
to be used as an indicator of protein abundance. Nevertheless, a score
of 0.4 or less correlated with open reading frames unlikely to be
expressed based upon codon usage; while a score of 0.7 correlated with
open reading frames likely to be expressed based upon codon usage. The
CAI for M. xanthus phrA in E. coli is 0.3, suggesting
that its abundance in E. coli would be similar to that of LacI
with a CAI of 0.296(19) . In comparison, analysis of the
published codon usage of E. coli phrA(39) using the
relative adaptiveness of codons in E. coli(19) gives
a CAI of 0.281 for E. coli phrA in E. coli. The
observation that, based upon amino acid similarity, the photolyase of M. xanthus belongs in a class containing none of the other
sequenced eubacterial photolyases was surprising. It suggests that the
divergence of the two classes of photolyase from a parent gene occurred
in the evolutionarily distant past. Both classes of photolyase are
found in the Archaea as well as the eubacteria (the photolyase of Halobacterium halobium belongs to the class containing
eubacterial and fungal photolyases; (4) and (33) ). In
all likelihood, the ``eukaryotic'' form of photolyase is more
common among the eubacteria than has been observed to date.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
U44437[GenBank].
Volume 271,
Number 11,
Issue of March 15, 1996 pp. 6252-6259
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Higher
Eukaryotes than
to Photolyases of Other Eubacteria (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
45,071). By comparison of the amino acid
and DNA sequences with those of other known photolyases, it has been
found that it is more similar to the deduced amino acid sequences of
the photolyases of ``higher'' eukaryotes than to the
photolyases of other eubacteria. Recombinant plasmids carrying M.
xanthus phrA rescue the photoreactivation activity of an
irradiated strain of Escherichia coli with a deletion in phrA. This rescue is light-dependent.
to 10
cells are formed in
which individual cells differentiate into spores. phrA is
linked to the chemotaxis genes of M. xanthus (the frz operon), but is transcribed in the opposite
orientation(5, 6) . The deduced amino acid sequence of
this photolyase is similar to the photolyases of other eubacteria in
the highly conserved carboxyl terminus domain. However, it is similar
to the photolyases of vertebrates and insects throughout the entire
protein(3) . Nevertheless, the gene is able to rescue
photoreactivation activity in a phrA mutant
of Escherichia coli. The classification of the M. xanthus photolyase with the photolyases of organisms in other kingdoms
rather than with photolyases of other eubacteria suggests that the
evolution of the two forms of photolyase is ancient.
Strains, Plasmids, and Growth Conditions
Strains
and plasmids are described in detail in Table 1. SY2 was a
generous gift from A. Yasui (Institute of Development, Aging and
Cancer, Tohoku University, Sendai, Japan). E. coli was grown
in LB medium(11) . M. xanthus was grown in CYE
medium(12) . Components of growth media were manufactured by
Difco. Salts were purchased from Fisher. Other, nonradioactive,
chemicals were purchased from Sigma.
Cloning phrA and Recombinant DNA
Standard
techniques were used for the construction of recombinant DNA,
restriction by endonucleases, and analysis by agarose gel
electrophoresis(13) . The subcloning of the region of DNA
encoding M. xanthus phrA is shown in Fig. 1. Plasmids
are described in Table 1.
Sequencing of DNA
Single-stranded DNA was purified
from M13KO7 lysates of JM107 carrying either pML118 or pMW119 as
described in the Promega Lab Manual (Promega, Madison, WI).
Single-stranded template DNA was sequenced using a modification of the
7-deaza-dGTP Sequenase kit from U. S. Biochemical
Corp. using [
S]deoxycytidine
5`-(
-thio)triphosphate (1000-1500 Ci/mmol, 12.5 mCi/ml,
DuPont NEN). In addition to the reagents supplied by the manufacturer
in the Sequenase kit, an extension mix consisting of
7-deaza-dATP, 7-deaza-dGTP, dCTP, and dTTP (180 µM each)
and 50 mM NaCl was used. The extension mix is equivalent to
the extension mix supplied by the manufacturer with the standard
Sequenase
kit except that the 7-deaza forms of dATP
and dGTP are substituted for the standard nucleotides. 1.0 µl of
extension mix was used with 1.5 µl of termination mix from the
7-deaza-dGTP Sequenase
kit. The idea to include both
7-deaza-dATP and 7-deaza-dGTP in the sequencing reaction came from the
catalog description of a sequencing kit manufactured by Pharmacia
Biotech Inc. that is not available in the United States. After the
termination reaction, terminal transferase (Promega) was added
according to the manufacturer's instructions with dATP at a final
concentration of 1 mM for 30 min at 37 °C(14) .
17-mer oligonucleotide primers were synthesized by the Barker Hall DNA
Facility on site. Primers were purified using Oligonucleotide
Purification Solution (O.P.S.) according to the manufacturer's
directions (Midland Certified Reagent Co., Midland, TX). Sequencing
products were separated on 0.4-mm-thick 40% formamide, 7 M urea polyacrylamide gels made with Long Ranger
gel solution for DNA sequencing (FMC BioProducts, Rockland, ME)
in 1
TBE (90 mM Tris, 90 mM borate, 2 mM Na
EDTA, nominal pH 8.3). Following electrophoresis,
gels were fixed in 20% ethanol, 10% glacial acetic acid for 15-30
min prior to being dried for autoradiography using BioMax MR film
(Eastman Kodak Co.)Analysis of DNA Sequence
The DNA Inspector IIe
(Textco, West Lebanon, NH) software for the Macintosh was used to
translate the DNA sequence into open reading frames. Protein data bases
were searched using the BLAST algorithm (15) with the BLOSUM62
substitution matrix for determining the degree of similarity between
two proteins(16, 17) through e-mail to NCBI
(blast@ncbi.nlm.nih.gov). MACAW (Multiple Alignment Construction
and Analysis Workbench, (18) ), obtained by ftp transfer of
public domain software from NCBI, was used to align overlapping DNA
sequences for both strands of the DNA.Determination of Codon Adaptation Index
The codon
adaptation index (CAI) (
)for open reading frames was
determined as described by Sharp and Li(19) . A compilation of
the codon usage of 22 protein coding regions of M. xanthus(20) was used to calculate the relative adaptiveness of
each codon (w): among synonymous codons, the number of times
that each is used is divided by the number of times that the optimum
codon is used. For example, GAU and GAC are synonymous for aspartate.
In the Shimkets's compilation, GAU is used 54 times while GAC is
used 319 times. The values for the relative adaptiveness of each codon
are therefore 0.17 and 1, respectively. The optimum synonymous codon
always has the value of 1. The CAI of the protein coding region is
equal to the product of the relative adaptiveness of each codon used in
the protein raised to the power of 1 divided by the number of codons in
the protein. For a protein of 500 amino acids, CAI = (w w
. . . w)
(see Equation 6 in (19) ).
Alignment of Similar Protein Sequences
MACAW (see
above) was used to align protein sequences identified by BLAST to be
similar to M. xanthus photolyase. Statistical parameters used
are described in figure legends and text. Further modifications of the
alignment were made by the researchers according to their best
judgment.Prediction of Protein Secondary Structure
The
program Peptidestructure (21) of the GCG (Genetics Computer
Group, Madison, Madison Wisconsin) analysis package was used to predict
the structure of E. coli and M. xanthus photolyases.Photoreactivation
Photoreactivation was assayed in
a manner similar to that described previously(2) . SY2 strains
were grown overnight under selection in LB medium containing
chloramphenicol (25 µg/ml) and ampicillin (100 µg/ml). The
overnight culture was diluted 1:100 into LB and shaken vigorously at 37
°C. Cells were harvested during exponential growth by
centrifugation at 3,000 g, at 4 °C, for 5 min.
Cells were resuspended at 10
cells/ml in M9
medium(11) . Aliquots (1.0 ml) of cells were placed in 50-mm
plastic tissue culture dishes (Falcon 3002). The dishes were placed in
a UV Stratalinker 1800 (Stratagene, La Jolla, CA),
the lids were removed, and the cells were irradiated with ultraviolet
light (254 nm) at the power setting described in the text. The cells
were further incubated for 1 h in darkness or exposed to ambient light
from fluorescent illumination on a laboratory bench to allow
photoreactivation to occur. After 1 h of incubation, cells were diluted
in M9 and plated on LB agar (1.5%). Plates were incubated at 37 °C
overnight in the dark. Colonies were counted to determine recovery from
UV irradiation.
Primer Extension and Identification of the Start Site for
Transcription
Total RNA was purified from M. xanthus DZF1 in exponential growth phase using 6 M guanidinium
isothiocyanate, 50 mM Tris, pH 6.8, 0.5% Sarkosyl, and 1
mM 
-mercaptoethanol(22) . Primer extension was
performed using an avian myeloblastosis virus reverse transcriptase
primer extension kit (Promega). The primer (GCTGGGCCAGCCGGGGA) is
complementary to the mRNA at positions 700-716 of Fig. 2.
In lieu of 5`-end-labeling of the primer, 6 µCi of
[S]deoxycytidine 5`-(
-thio)triphosphate
were added to the extension reaction.
Sequencing and Identification of Open Reading
Frames
DNA was sequenced and open reading frames were identified
as described under ``Materials and Methods.'' The sequence
shown in Fig. 2has a G+C content of 68 mol%, which is
consistent with previous observations of 67-71 mol% for M.
xanthus DNA (for a review, see (20) ). Organisms with this
level of G+C in their genomes tend to use codons that have a G or
C residue in the third position(23) . Indeed, Shimkets's (20) analysis of 22 protein coding regions of M. xanthus demonstrated that 91% of the codons contain a G or C residue in
the third position. The first position is 70% G+C, the second
position is 47% G+C. These observations make it possible to
predict which open reading frames are likely to be translated by
determining codon usage and G+C content at the three positions. An
additional analysis, Codon Adaptation Index, has been described for
determining the likelihood that a protein is highly
expressed(19, 24) . The codon usage table compiled by
Shimkets (20) was used to determine the relative adaptiveness
of each codon for M. xanthus (Table 2). The eight open
reading frames of 
100 amino acids were analyzed for G+C
content at each position of the codon, and the CAI was determined (Table 3). The analyses suggest that only one of the open reading
frames is likely to be a protein coding region. The amino acid sequence
for this open reading frame was compared to the protein data banks
using the BLAST algorithm with the BLOSUM62 substitution matrix (15, 16) as described under ``Materials and
Methods.'' Open reading frame 7 (ORF7) has strong similarity to
photolyases of a number of organisms with greater similarity to the
photolyases of Methanobacterium, Vertebrata, and Insecta than
with Eubacteria (Table 4).
Demonstration of Photoreactivation Activity in
phrA
To demonstrate that ORF7 encoded a
photolyase activity, photoreactivation assays were performed as
described under ``Materials and Methods.'' E. coli cells were irradiated with enough energy to kill E. coli Harboring Recombinant Plasmids Carrying
the phrA Gene of M. xanthus
99.99% of the
cells. For SY2, SY2/pUC118, and SY2/pUC119, this was 4400 µJ.
SY2/pML118 was irradiated at 4200 µJ; SY2/pMW119 and SY2/pBB12 were
irradiated at 4300 µJ. In our hands, there is some residual
photoreactivation activity in SY2: cells recovering from irradiation in
the dark gave 0.01% of the number of colonies obtained from
unirradiated controls, cells recovering in the light gave 0.1% of the
number of colonies of unirradiated controls. Thus, there was a 10-fold
increase in the number of survivors through recovery in the light
compared to the dark. A 10-fold increase in survivors through recovery
in the light was also seen for SY2/pUC118 and SY2/pUC119 (no phrA DNA). SY2/pBB12 demonstrated a 10,000-fold increase in survivors
through recovery in the light (0.01% viable through recovery in the
dark, 95% viable through recovery in the light). SY2/pML118
demonstrated a 1000-fold increase in survivors through recovery in the
light (0.01% viable through recovery in the dark, 10% viable after
recovery in the light). SY2/pMW119 demonstrated a 100-fold increase in
survivors through recovery in the light (0.01% viable through recovery
in the dark, 1% viable after recovery in the light). These results
demonstrate that ORF7 not only has strong sequence similarity to known
photolyases, but that plasmids carrying ORF7 allow photoreactivation in
the heterologous E. coli system. Henceforward, ORF7 will be
designated phrA, a photolyase gene of M. xanthus.Identification of the phrA Promoter and Analysis of
Characteristics of the DNA and Amino Acid Sequences
The open
reading frame with similarity to photolyases begins at nucleotide 419
in Fig. 2. Primer extension of total RNA from M. xanthus shows that the start site for the phrA mRNA is at
nucleotide 486 (data not shown) and lies within the photolyase open
reading frame (Fig. 2). A promoter that is consistent with the
consensus sequence and the proper spacing for promoters of M. xanthus was inferred to have a -35
region beginning at nucleotide 452 and a -10 region beginning at
nucleotide 477 (Table 5). The first translation start codon
accompanied by a ribosome binding site occurs in the cluster MMDM
beginning at nucleotide 619. The putative ribosome binding site, GGAGG,
begins at nucleotide 613 (Fig. 2). This ribosome binding site is
consistent with the known 3` sequence of M. xanthus 16 S RNA
(3`HO-UCUUUCCUCCACUA; (34) ) and is the same as that suggested
for M. xanthus dsg(35) . Consensus spacing of ribosome
binding sites with the start of translation suggests that the start of
translation would begin with the second AUG after the ribosome binding
site (within approximately 10 bases of the ribosome binding site; (36) ). These assumptions predict that M. xanthus photolyase would have a length of 401 amino acids and be 45,071
daltons in size.
1) are
denoted by a white letter on a gray background. The
putative first translated methionine in M. xanthus photolyase
is underlined. The structure of E. coli is
from(37) : open rectangles are -helices, black rectangles are -sheets, hatched rectangles are 3 helices. Residues involved in FAD binding are
marked with a ˆ below the residue. > indicates a residue
involved in binding FAD through H
O. 
denotes residues
that form hydrogen bonds with MTHF. 
denotes residues which
interact with MTHF through HO.
) to UV irradiation (as demonstrated by
the lower dosage of UV radiation need to kill 99.99% of the cells) when
expressing M. xanthus photolyase is consistent with previous
observations(38) . The difference in the efficiency of
photoreactivation among the three plasmids may be due to the
orientation of phrA relative to the lacZ promoter of
the vectors; the orientation of ORF7 in pMW119 is such that an
antisense mRNA could be transcribed from the lacZ promoter,
leading to a reduction in the effective expression of the protein.
Alternatively, since the
promoter identified by
inspection of the DNA sequence is unlikely to function efficiently in E. coli, it may be that increased efficiency of
photoreactivation by the pBB12 and pML118 clones is due to
transcription of phrA from the lacZ promoter of the
vector (Fig. 1).
-helices and -sheets in E. coli photolyase (37) and suggested that the pattern is
conserved in M. xanthus photolyase (data not shown). The
program did less well in predicting the structure of the carboxyl
terminus of E. coli photolyase; therefore, it is not possible
to predict the degree of conservation of structure in this region of
the protein. However, the greater degree of conservation of amino acids
in this region suggests that structure is likely to be conserved here
as well. Indeed, there is homology among the DNAs in this region among
the photolyases most closely related to that of M. xanthus (Fig. 4).-helices and -sheets is conserved in the untranslated region
(GCG Peptidestructure (see above)), further suggesting that this region
was recently under selection.
)
We acknowledge Dr. Akira Yasui for encouraging
correspondence, and we thank him for sharing the sequence of M.
thermoautotrophicum prior to publication. We thank Mike Lai, Ann
Tang, and Cannit Tsai for their work on the sequencing of phrA.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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