(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 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.
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 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.
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 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 H
O.
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) 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).
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 -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).
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
-helices and
-sheets is conserved in the untranslated region
(GCG Peptidestructure (see above)), further suggesting that this region
was recently under selection.
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].