(Received for publication, May 15, 1995; and in revised form, July 11, 1995)
From the
The Dps family of proteins are a diverse group of bacterial
stress-inducible polypeptides that bind DNA and likely confer
resistance to peroxide damage during periods of oxidative stress and
long term nutrient limitation. Some members of the Dps protein family
have been shown to form large (150-kDa), hexameric complexes that
bind chromosomal DNA with little sequence specificity. In this paper we
report the nucleotide sequence of the dpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps
homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA protein revealed that a carboxyl-terminal domain of the
protein was >60% homologous to the COOH-terminal half of
bacterioferritin. Other known Dps family members lack such high
similarity to the bacterioferritins. Purification and spectroscopic
analysis of the Synechococcus sp. DpsA protein complex
revealed that the complex contains heme and has a weak catalase
activity in vitro. Activity staining of nondenaturing
polyacrylamide gels showed that the protein complex comigrated with
both the heme and the catalase activity, and O
evolution
measurements yielded a maximal specific activity of 1.7 µmol of
H
O
consumed/µg of protein
min
. We speculate that the protein may have a
peroxide-consuming mechanism located on the chromosomal DNA, and we
also suggest that this activity may be a necessary feature to handle
the endogenous oxidative stresses associated with oxygenic
photosynthesis. Last, the evolutionary link between the Dps protein
family and the bacterioferritins is discussed.
The Escherichia coli Dps protein(1) , also
known as the PexB protein(2) , was first identified as a
DNA-binding polypeptide that accumulated to high levels during
stationary phase, and analysis of E. coli mutants lacking Dps
function suggested strongly that the function of the protein is to
protect DNA from oxidative damage(1) . Additional work has
established that the E. coli Dps/PexB protein accumulates
under a number of different regimes of oxidative stress and nutrient
limitation(2) . The expression of dps/pexB during
oxidative stress in the growth phase is dependent on positive
regulation by OxyR(3) , although under stationary phase, dps/pexB transcription is dependent on the alternative
factor RpoS and the DNA-binding protein IHF(3, 4) .
Ultrastructural studies have also shown that the protein forms a
complex composed of hexagonal, hexameric rings both in the presence and
absence of DNA(1) . In a recent publication(5) , we
described the purification and characterization of a 22-kDa polypeptide
from nitrogen-starved Synechococcus sp. PCC7942 that is
structurally and functionally similar to the E. coli Dps
protein. From our studies, the Synechococcus sp. strain
PCC7942 Dps homolog (henceforth named DpsA) was first purified from
nitrogen-limited cells as a large polypeptide having an approximate
molecular mass of 150 kDa; subsequent peptide sequencing and
electrophoretic studies under denaturing and nondenaturing conditions
indicate that this polypeptide is a complex of the DpsA protein
containing single stranded DNA(5) . Denaturing PAGE (
)indicated that the DpsA monomer had an apparent molecular
mass of 22 kDa(5) . Due to the chemical properties of this
polypeptide and its accumulation in nutrient-limited and stationary
phase cells, we suggested that this polypeptide serves a similar
function to that proposed for the Dps protein in E.
coli(1, 5) . Many diverse bacteria also express
Dps homologs, although only the E. coli, Bacillus subtilis,
and Synechococcus sp. proteins have been well characterized
with respect to DNA-binding activity and accumulation under stress
conditions(1, 5, 6) . (
)In this
paper, we have cloned and sequenced the Synechococcus sp. dpsA gene, and the deduced amino acid sequence suggested that
the DpsA protein is a hemoprotein due to the presence of a
COOH-terminal domain homologous to the COOH termini of
bacterioferritins(8, 9) . Other members of the Dps
protein family do not exhibit this extensive degree of similarity to
bacterioferritin. Characterization of the purified DpsA protein
revealed that the complex exhibits a weak catalase activity, and
spectroscopic studies revealed the presence of heme in the complex.
This raises the possibility that a heme-dependent enzymatic activity
plays a role in protecting DNA from peroxide damage in oxygenic
phototrophs. Furthermore, the sequence similarity between the DpsA
protein and the bacterioferritins suggests that they share a common
evolutionary origin.
Figure 1: Synthesis of the 69-nucleotide double-stranded dpsA probe by degenerate PCR. From both the N-terminal peptide sequence of the Synechococcus sp. DpsA protein (5) and the consensus peptide sequence for other Dps family members(2) , two degenerate oligonucleotides were synthesized that amplified a 69-base pair fragment encoding residues 43-65 of the DpsA protein (bottom). The asterisk designates the glutamic acid residue that later proved to be an aspartic acid residue following complete sequencing of the dpsA gene.
Figure 2:
Nucleotide sequence of the Synechococcus sp. The PCC7942 dpsA gene and the
deduced primary structure of its product are shown. Nucleotide
sequences underlined include the putative ribosome binding
site and rho-independent transcriptional terminator. Peptide sequences underlined correspond to sequences obtained by automated Edman
degradation of NH-terminal and endoproteinase Lys-C
fragments of the purified protein.
Screening a ZAP Synechococcus genomic library
with the 69-base pair PCR product encoding amino acid residues
43-65 of the DpsA protein resulted in the retrieval of several
ZAP clones covering the dpsA gene. DNA sequencing
revealed a 528-nucleotide open reading frame encoding all of the
peptide sequences determined by Edman degradation of the purified
protein (Fig. 2). The molecular mass of the deduced DpsA protein
was 19,692 Da, slightly smaller than the estimates from denaturing
PAGE; the isoelectric point was calculated to be 5.15. Lipman and
Pearson (19) protein sequence analysis confirmed that the DpsA
protein is homologous to other members of the Dps protein family (Fig. 3, residues in blue). Other members of this small
family include the B. subtilis MrgA protein (6, 7) an antigen from Treponema sp.(20) , a cold-shock-inducible protein from the
filamentous cyanobacterium, Anabaena variabilis(
)and a putative product of an open reading frame from Streptomyces aureofaciens(21) . Comparison of the DpsA
protein to other members of the Dps family indicated that the protein
has 19% identity, 38% similarity to the E. coli Dps/PexB
protein and 20% identity, 40% similarity to the homolog from A.
variabilis.
All the Dps polypeptides are most likely
stress-inducible DNA-binding proteins as suggested by the DNA-binding
activity demonstrated for the E. coli, B. subtilis, and Synechococcus sp.
homologs(1, 5, 6) .
What was
unexpected was the presence in the Synechococcus sp. DpsA
polypeptide of a COOH-terminal domain highly similar in sequence to a
COOH-terminal sequence of bacterioferritin (Fig. 4, (8) ). Comparison to the Azotobacter vinelandii bacterioferritin (8) exhibits 55% similarity (27/49
residues starting with Met-116 of the DpsA protein). Additionally,
there is a detectable, albeit lower, degree of sequence similarity
between the DpsA protein and bacterioferritin NH
termini (Fig. 3, residues in red). Comparing the COOH termini,
virtually all the residues that represent the bacterioferritin
consensus in this sequence are conserved (Fig. 4), and over the
entire DpsA polypeptide, 55% of the bacterioferritin consensus sequence
is conserved (Table 1). Overall, the DpsA protein is 39% similar
to the A. vinelandii bacterioferritin (Fig. 4). It
should be pointed out that the other members of the Dps protein family
do not exhibit this high degree of sequence similarity to the
bacterioferritins (Fig. 3). However, alignment of the
bacterioferritin consensus sequence (7, 8, 9) to the Dps protein family indicates
shared residues across both groups, suggestive of a common evolutionary
origin (Fig. 3; residues of the bacterioferritin consensus shown
in blue). Furthermore, individual members of the Dps family
also yield similarities to the bacterioferritin consensus (Fig. 3, residues in red). Also included in this
analysis are sequences of two proteins from Helicobacter pylori and Hemophilus ducreyi that were retrieved from the
GenBank
data base but had not been previously assigned to
the Dps family (Fig. 3).
The overall percentage of
similarity of all these proteins to the bacterioferritin consensus is
summarized in Table 1.
Figure 3:
Comparison of the DpsA protein to the
bacterioferritin (bottom lines, italics) and Dps family (top lines, italics) consensus sequences. Residues of the
shared Dps family signature sequence (1) are marked in blue; those shared with the bacterioferritin consensus (7) are shown in red. Those residues represented in
both the Dps and bacterioferritin consensus are indicated in blue in the bacterioferritin consensus sequence (italics, bottomlines). Dps/PexB, E. coli Dps protein; S. aure, S. aureofaciens Dps
homolog; A. var, A. variabilis Dps homolog; MrgA, B. subtilis MrgA protein; TYF1 An, Dps
homolog (TYF1 antigen) from Treponema sp.; H.
ducreyi, putative neutrophil activating protein from H.
ducreyi; H. pylori, putative pilin precursor
protein
; Bfr, A. vinelandii bacterioferritin. The H. ducreyi and H. pylori proteins have not been previously assigned to the Dps protein
family. The asterisk at Met-116 of the DpsA sequence indicates
the start of the region of strong homology to Azotobacter bacterioferritin. Overall, 55% of the bacterioferritin consensus
sequence is represented in DpsA (see Table 1for
summary).
Figure 4: Comparison of the DpsA primary structure to A. vinelandii bacterioferritin. The residues similar to the domain covering residues 86-135 of A. vinelandii bacterioferritin are indicated in boldface. Those positions corresponding to the bacterioferritin consensus sequence (7) are indicated with plus (+) symbols.
The sequence similarity of DpsA to A. vinelandii bacterioferritin ( Fig. 3and Fig. 4) led us to determine whether the Synechococcus sp. DpsA protein could bind heme, as has been shown for the
bacterioferritin proteins(7, 8, 22) . Indeed, purification of the DpsA protein complex yielded an
orange preparation having spectroscopic properties of bound heme;
UV/visible absorption spectra yielded a complex spectrum having
putative Soret absorbance maxima at 352-410 nm and an additional
peak at 552 nm (Fig. 5A). Furthermore, treatment of the
material with pyridine at alkaline pH yielded a pyridine hemochrome
derivative having absorbance maxima around 410 and 550 nm (Fig. 5B). Such absorbance bands are diagnostic for
heme and are likely indicative of protoporphyrin IX(17) . Based
on the molar absorptivity of proto IX, we estimate there is
approximately 1 mol of heme bound per mol of DpsA hexamer. This low
heme content may indicate that the complex lost heme during
purification; this is not very surprising, given that the purification
scheme depends on gel filtration in 4 M urea and elution from
DEAE-cellulose at 1.5 M NaCl. Preparations of the DpsA complex
reported initially (5) did not have an obvious color, most
likely because the complex was dissolved in buffers containing
-mercaptoethanol. Addition of reducing agents allowed for the
complex to be dissociated more easily into the 20-22-kDa DpsA
monomer (5) , and such treatment probably aided in extracting
any prosthetic groups from the protein complex.
Figure 5: A, visible absorption spectrum of the DpsA protein complex; B, visible absorption spectrum of the pyridine hemochrome derivative of the DpsA preparation.
Since the Dps
proteins are associated with resistance to peroxide stress, these data
raised the possibility that the prosthetic group could serve to consume
peroxide by a heme-dependent peroxidase or catalase mechanism. Indeed,
testing the complex for catalase activity yielded modest rates of
oxygen evolution when assayed in the presence of 9 mM
HO
(Fig. 6); however, the complex did
not yield detectable peroxidase activity in the presence of substrates
such as guaiacol (2-methoxyphenol). More detailed analysis revealed
that the activity had an apparent K
of 11
mM, and a V
of 1.7 µmol of
H
O
consumed µg protein
min
. Additionally, activity staining of gels
in which the purified DpsA protein complex had been electrophoresed
under nondenaturing conditions revealed that the complex retained
catalase activity (Fig. 7); the zone of clearing associated with
catalase activity (laneC) identified the position in
the gel to which both the orange color (laneA) and
the immunoreactive Dps protein complex comigrated (lanesB and D). Thus, the gel data demonstrate that under
conditions of low denaturation, heme remains bound to the protein and
the protein complex retains a low level of catalase activity. These
data argue against the possibility that a minor contaminating catalase
activity copurified with the DpsA protein complex.
Figure 6:
Catalase activity of the DpsA protein
complex. Ten micrograms of protein were assayed in 9 mM HO
; oxygen production was monitored by a
Clark-type electrode. The protein complex was added at the time point
indicated by the arrow, and the reaction was stopped by the
addition of sodium azide to 6 mM. Analysis of the pen
deflection yielded a O
evolution rate of 410 nmol/µg
protein/min (corresponding to 820 nmol of H
O
consumed/µg protein/min).
Figure 7: Electrophoresis of the DpsA protein complex. Left to right, photograph of the unstained gel after nondenaturing PAGE showing the orange-colored DpsA complex (laneA); identical gel following silver staining (laneB); parallel sample stained for catalase activity (laneC); parallel sample immunoblotted and probed with the DpsA antibody (laneD). The darkerregion in the middle of the zone of catalase activity is contributed by the orange color in the sample. The arrow next to the rightpanel indicates the migration of the prestained 97-kDa molecular mass marker in this PAGE system.
In this paper we report that the Dps homolog from Synechococcus sp. PCC7942 has a weak catalase activity in
vitro. This activity is consistent with the proposal that the Dps
proteins function to protect the chromosomal DNA from peroxide
damage(1) . Of course, it is not possible at this time to state
for certain whether this catalase activity is present in vivo,
but it is attractive to speculate that the bound heme is involved in a
peroxide-consuming mechanism located at the chromosome. A problem with
this proposal is that the activity of the purified complex is very low,
yielding a K that is likely not physiological.
However, it is possible that this low activity reflects the loss of
heme during purification, as suggested by the low heme content
determined by spectroscopy. Additionally, the DpsA complex has been
purified as a 150-kDa soluble protein-DNA-heme complex that is quite
different in organization from the high molecular mass supramolecular
arrays of the E. coli Dps protein complexed to linear DNA seen
by electron microscopy(1) . Perhaps similar large arrays of the Synechococcus sp. DpsA protein complexed to large chromosomal
DNA fragments are required for maximum activity.
We should stress
that other members of the Dps protein family do not exhibit the
similarity in sequence to the bacterioferritins exhibited by the DpsA
protein (Fig. 3). Thus, other members of the Dps family may lack
this heme-binding function. If so, perhaps the endogenous oxidative
stresses associated with photosynthetic metabolism in Synechococcus sp. require additional function(s) provided by the heme-binding
structure. Additionally, since both the DpsA protein and
bacterioferritins form extremely stable oligomeric
complexes(1, 5) , perhaps this similar
domain is also involved in forming or stabilizing these quaternary
structures. Knowing that the DpsA protein binds heme may help explain
why the protein accumulates under all nutrient stresses tested except iron limitation (5) . It is possible that in Synechococcus sp., the functional assembly and stability of
the protein-DNA-heme complex is dependent on the presence of iron in
the porphyrin ring. Alternatively, iron limitation may alter dpsA expression at the transcriptional level.
The data presented
here also suggest strongly that the DpsA protein is a divergent member
of the bacterioferritin/ferritin superfamily (8) . Previous studies have yielded an evolutionary link between
bacterioferritins and eukaryotic ferritins(8) , and thus we
propose that the Dps protein family shares a common evolutionary origin
with this group. We argue that the Dps proteins may have evolved
initially as heme-binding or metal-binding complexes that later
acquired DNA-binding activity. However, the other members of the Dps
family lack the strong similarity to bacterioferritin, and to date no
one has reported a heme-binding activity associated with other Dps
proteins. Examining the bacterioferritin and Dps consensus sequences
does reveal some sequence conservation across the Dps family (Fig. 3, Table 1), and this includes bacteria of divergent
lineage (e.g.Streptomyces and E. coli).
Making the assumption that the bound heme has some enzymatic function in vivo, we suggest that among the heterotrophic bacteria, the
heme-binding domain became dispensable, yet was retained in a
cyanobacterium whose obligate oxygenic metabolism would likely yield
greater demands for oxidative protection. Extensive sequence analysis
and biochemical studies of Dps family members from representative
phylogenetic groups will be necessary to test this hypothesis.
We
also note that the Dps protein family may include other gene products
that are listed in the available data bases and may be assigned other
putative functions. A polypeptide described as a neutrophil-activating
protein from H. pylori (25) having properties of a novel
bacterioferritin was retrieved from our search of the GenBank data base with the dpsA sequence, and such comparisons
indicate that the H. pylori protein has the peptide signature
diagnostic for the Dps family (see Fig. 3). Additionally, a
protein described as a pilin precursor from H. ducreyi (26)
similarly has the Dps signature sequence (Fig. 3). Determining
the precise roles for these polypeptides awaits further investigation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U19762[GenBank].