(Received for publication, February 24, 1995; and in revised form, July 5, 1995)
From the
Both site-directed and spontaneous mutagenesis have been used to
investigate the role of the cis-acting regulatory region
between -92 and -1 base pair (bp) of the puc operon of Rhodobacter sphaeroides. The DNA sequence from
-84 to -66 bp upstream of the 5` end of the start site of puc operon transcription is essential for normal puc operon expression. This regulatory effect was exerted irrespective
of the presence or absence of additional upstream regulatory sequences
extending from -629 to -93 bp. It is likely that this
region is involved in activator binding. Additionally, two regions of
dyad symmetry centered at -42 and -17 bp are shown to be
involved in oxygen repression of puc operon expression.
Mutations within these regions of dyad symmetry were further subdivided
on the basis of whether or not the upstream regulatory region was
required to observe the mutant phenotype. Based upon these observations
we conclude that these regions of dyad symmetry possessing the motif
TGT-N-ACA (where N represents any nucleotide) are involved
in repressor binding with the puc operon promoter overlapping
each of these dyad symmetries.
The puc operon of Rhodobacter sphaeroides consists of two structural genes, pucB and pucA,
encoding the - and
-polypeptides, respectively, of the
B800-850 light harvesting complex and additional downstream open
reading frame(s) that encode gene product(s) involved in the
post-transcriptional control of expression and assembly of the
-
and
-polypeptides to form the B800-850
complex(1, 2) . In a related bacterium, Rhodobacter capsulatus, the puc operon has been
reported to have a similar although not identical organization (3, 4) . Formation of the B800-850 complex is
variable with respect to the fixed photosynthetic unit. Together with
the fixed photosynthetic unit the B800-850 complex has been
referred to as the variable photosynthetic unit, which can comprise
over 50% of the protein found in the intracytoplasmic
membrane(5, 6, 7, 8, 9) .
Intracytoplasmic membrane formation is represented as a series of
invaginations arising from and continuous with the cytoplasmic membrane
following removal or lowering of the partial pressure of O
below threshold levels, approximately 2.5% relative to
air(10) .
The puc operon gives rise to two (0.5-
and 2.3-kilobase) transcripts, having a single transcription start
site, both of which are severely affected by the presence or absence of
O, and in the absence of O
the relative levels
of each transcript are further modulated by light
intensity(1, 11) . We have subdivided the 629-bp (
)puc DNA upstream of the 5`-end (+1) of the puc transcripts into two functionally separable cis-acting domains, the upstream regulatory sequence (URS)
(-629 to -93) involved in enhanced control of operon
transcription by oxygen and light, and the downstream regulatory
sequence (DRS) (-92 to -1) primarily involved in basal
level aerobic transcription as well as anaerobic regulation of
transcription(12, 13) . Interaction or communication
between these two cis-acting regions was demonstrated based
upon the transcriptional response to oxygen and light and the silencing
of point mutations within the DRS when these two domains are
uncoupled(12) . A region extending from -139 to -93
bp containing overlapping FNR- and IHF-binding sequences separates
these two cis-acting regions and is suggested to play a role
in mediating this interaction(12, 13, 14) .
The IHF-binding sequence is involved in repression of puc operon transcription by oxygen as well as modulation of the
transcription levels by incident light intensity(14) . Recently
the gene encoding an FNR homologue in R. sphaeroides has been
identified in our laboratory. (
)
Recently Eraso and Kaplan (15) reported a new gene locus in R. sphaeroides, prrA, which was found to be involved in positive regulation of
photosynthesis gene expression in response to anaerobiosis. The
NH-terminal portion of PrrA showed clear similarity to the
highly conserved amino termini of response regulators of two-component
regulatory systems mediating signal transduction in prokaryotes. PrrA
was shown to act (directly or indirectly) at the level of the DRS of
the puc regulatory region. In R. capsulatus a gene
designated regA was reported(16) , to which prrA shows a high similarity in both DNA and amino acid sequences. In
the present studies, both in vitro and in vivo mutagenesis were employed to investigate the role of the DRS in puc operon expression. As a result, the DRS was itself
subdivided into several regulatory regions. One of these domains is
similar to those reported to be involved in factor-dependent
binding(17) . A model is presented that describes the role(s)
for each of these regions within the DRS in the regulation of puc operon expression.
Escherichia coli strains JM109, DH5phe, TG1, or S17-1 were
grown at 37 °C in Luria medium(20) . Ampicillin,
tetracycline, kanamycin, streptomycin, and spectinomycin (final
concentrations, 50, 20, 25, 50, and 50 µg/ml, respectively) were
added to the growth medium for E. coli strains carrying
plasmids encoding these drug resistance genes. Plasmids pUC19, pBS,
pSL301, pRS415(21) , and pLV106 (12) were used for
cloning.
DNA sequence analysis was performed with phage M13 clones as described previously (11, 18) or with an ABI 373A automatic DNA sequencer (Applied Biosystems Inc., Foster City, CA) at the DNA Core Facility of the Department of Microbiology and Molecular Genetics (University of Texas Health Science Center, Houston, TX).
Figure 1: A, restriction map of the puc upstream DNA. +1 is the 5`-end of the puc-specific transcripts. The DNA region corresponding to the sequence shown in B is underlined. B, DNA sequence from -148 to +1. An IHF-binding sequence (b) overlapping a putative consensus sequence for FNR (a) is followed by the putative activator-binding site(s) (c). The two regions of dyad symmetry are distal (d) and proximal (e).
In
the first constructions, two consecutive bases, GG(-83,
-82) or GC(-68, -67) were changed to TT and TA,
respectively. Each of the altered DNA fragments corresponding to the
DRS (-92 to -1) with or without the URS (-629 to
-93) were transcriptionally fused to lacZ, each fusion
construct was mobilized into R. sphaeroides 2.4.1, and
-galactosidase activities were determined under the conditions
described.
From the data presented in Fig. 2it is obvious that the changes at -68 and -67 had a severalfold effect upon puc operon expression in both the presence and absence of the URS. Further, and most importantly when compared with wild type, the effect was observed regardless of the environmental conditions under which puc operon expression was measured. On the other hand, mutations at -83 and -82 showed little alteration in puc expression in the presence of the URS and little if any change in the absence of the URS.
Figure 2:
-galactosidase activities associated
with transcriptional fusions of the lacZ gene to puc upstream DNA containing either wild type or mutated forms of the
putative activator-binding sequence within the DRS. The puc DNA region corresponding to the 19 bp from -84 to -66
is underlinedbelow the restriction map and also
shown as a thickline for each puc::lacZ fusion. Mutated bases are denoted with asterisks. The puc::lacZ fusion constructs were present in trans in R. sphaeroides 2.4.1 grown chemoheterotrophically (30%
O
, 1% CO
, 69% N
) or anaerobically
in the dark with Me
SO. Photoheterotrophic cultures grown at
10 W/m
were harvested between 40 and 60 Klett units (1
Klett unit =
0.75
10
cells). Deviations
for
-galactosidase measurements are shown in parentheses.
A third mutation, deleting three
helical turns, i.e. 32 bp between -87 and -56 as
depicted in Fig. 3and designated c, was introduced in the
DRS. This deletion encompasses the region of
TGGC-N
-TCGCA
as
well as 3 and 10 bp upstream and downstream, respectively. The mutated
DRS was transcriptionally fused to lacZ in the presence and
absence of the URS and
-galactosidase activities determined as
described in the legend to Fig. 4. The removal of the DNA
sequence extending from -87 to -56 resulted in a
3-8-fold decrease in puc operon expression under the
conditions employed in both the presence and absence of the URS, when
compared with the wild type construction.
Figure 3:
A, the puc DNA sequence extending
from -93 to +1 illustrating the region mutated through
oligonucleotide-directed in vitro mutagenesis. denotes
deletion of the area confined by the bracketsabove the sequence. TGT (-52 to -50) and ACA
(-37 to -35) were mutated to GCG and CGC, respectively. B, the puc DNA sequence extending from -93 to
+1 illustrating the DNA region mutated by selection of regulatory
mutations employing the puc::aph fusion. cis mutations at -36 and -11 are shown below the
DNA sequence together with the previously described -26 and
-12 mutations(12) . Duplication of the 12-bp DNA sequence
from -34 to -23 is illustrated above the region of
duplication.
Figure 4:
-galactosidase activities associated
with transcriptional fusions of the lacZ gene to the puc upstream DNA containing wild type and various mutations associated
with the putative activator(s)-binding region and the regions of dyad
symmetry as described in Fig. 3. Each mutation can be identified
by noting the precise change to the immediateright of each plasmid designation. The solidlines indicate the presence of either the entire 629-bp upstream
sequence or the 92-bp upstream sequence. The puc::lacZ fusion
constructs were present in trans in R. sphaeroides 2.4.1 grown chemoheterotrophically (30% O
, 1%
CO
, 69% N
) or anaerobically in the dark with
Me
SO. Photoheterotrophic cultures at 10 W/m
were harvested between 20 and 30 Klett units. Deviations of the
-galactosidase activities are shown in parentheses. n.d., results not determined here.
One further point is worth
noting regarding the level of derepression when puc operon
expression is measured in cells growing aerobically and under
dark/MeSO conditions. When the URS is present, both the
c mutation and the mutations at -68 and -67 still show
an approximate 6-fold derepression, which is about 30% of that observed
for the corresponding wild type constructions. Thus, despite the
overall damaging effect upon puc operon expression, these
mutations largely retain the ability of the puc operon to
respond to the removal of O
.
Because these mutations mapped to one of the two
regions of dyad symmetry (Fig. 1B) a similar approach (12, 13) to the isolation of additional mutants
displaying increased resistance to Km was undertaken using even higher
concentrations of Km (40 and 80 µg/ml). Four independent isolates
of the same cis-acting mutation at -11 (C T) were
isolated at a Km concentration of 40 µg/ml (Fig. 3). At 80
µg/ml Km two additional mutations were isolated: the same at
-11 (C
T) as above and a duplication of the 12-bp DNA
sequence between -34 and -23 (Fig. 3). The 12-bp
duplication includes the left half of the proximal region of dyad
symmetry plus the gap sequence between the two regions of dyad symmetry (Fig. 3).
Another approach was employed to isolate a
different mutation at -36 (C T) (see Fig. 3). In
this case increased resistance to Km was selected in cells growing at
100 W/m
light intensity. Normally, these cells containing
the puc::aph fusion (12, 13) are resistant to
up to 2.5 µg/ml Km. At 3 W/m
these same cells are
resistant to 25 µg/ml Km. Thus, mutants resistant to 100 µg/ml
Km at 100 W/m
were isolated. Only one cis-acting
mutation (at -36) was obtained, and the remainder were shown to
be trans-acting mutations, which are not described here.
All of these mutations residing in the DRS were transcriptionally
fused to lacZ in the presence or absence of the URS and
-galactosidase activities measured under various conditions of
growth as depicted in Fig. 4. Upon examination of the results,
several points are noteworthy. As expected for the mutations at
-11 and the duplication of the sequence from -34 to
-23, the level of LacZ expression in the presence of O
is greater,
5-6 fold, than the corresponding wild
type. What was unexpected is that the mutation at - 36 yielded
LacZ activity 5-fold greater than wild type in the presence of
O
, although it was selected for in response to altered
expression in the presence of high light under anaerobic conditions.
However, unlike the former two mutations, the change at -36 did
lead to increased expression either under conditions of anaerobic dark
or anaerobic light growth, when compared with the wild type. When the
DRS was uncoupled from the URS, each of these three mutations resulted
in different levels of expression of
-galactosidase. The mutation
at -36 was essentially silent, and the duplication showed
decreased expression. However, the mutation at -11 showed
increased LacZ expression in the presence or absence of O
.
In the complete absence of the distal region of dyad symmetry
(d) the level of
-galactosidase in the absence of the URS is
near background. Even in the presence of the URS, LacZ levels are low,
and there appears to be no increased expression upon removal of
O
.
Also evident is the fact that changes at positions
-52 to -50 and -37 to -35 lead to increased
LacZ activity in the presence of O, and in the case of
-52 to -50 increased expression in the absence of O
is observed when the URS is present. In the absence of the URS,
only the changes at -37 to -35 show increased
-galactosidase in the presence or absence of O
,
whereas the changes at -52 to -50 are silent when these
mutations are compared with the wild type. However, both sets of
changes continue to show derepression under an aerobic to anaerobic
transition. Thus, it appears that both regions of dyad symmetry play a
role in the repression of puc operon expression in the
presence of O
as well as in derepressed expression of the puc operon in response to the removal of O
. Also,
mutations localized to these regions of dyad symmetry respond
differently, depending upon whether or not the DRS and URS are coupled
or uncoupled.
The puc operon upstream DNA between the XmaIII(-92) and the HaeIII(-57)
restriction sites immediately downstream of the overlapping FNR- and
IHF-binding sequences was shown previously to play an important role in
the expression of the puc operon under both aerobic and
photoheterotrophic conditions when the DRS was uncoupled from the URS (12) . For the purposes of this discussion expression of lacZ is considered to be a relative measure of puc operon transcription. The results reported here support further
the importance of this DNA sequence centered at approximately
-75. We propose that this region serves as a binding site for a
positively acting transcriptional regulator(14) . The fact that
the presence of this region results in an increased expression of the puc operon whether or not O is present would be
anticipated for a generalized transcriptional activator. The mutation
at -68 and -67 appears to have a lesser effect in the
presence of oxygen than in its absence. This may reflect, as discussed
below, the ability of the hypothetical activator to contact RNA
polymerase whose promoter binding ability would be strongly affected by
the presence of repressor when O
is present.
Although
the results of this study were focused on the DRS, the question
naturally arises as to the role or roles of the URS since there are
absolute differences in the expression of the puc operon
reported here and elsewhere (12, 13, 14, 15) dependent upon the
presence or absence of the URS. One possibility is that a second
promoter is present in the URS in addition to a promoter within the
DRS. There are several observations arguing against that possibility.
First and foremost all attempts to detect puc operon
transcripts or 5` ends derived from DNA sequences upstream of the
reported unique 5` end (1) have proven unsuccessful despite
the method of RNA preparation. Second, the response regulator PrrA
having a major effect upon puc operon expression has been
interpreted to exert its effect upon puc operon expression
through the DRS. Third, the silencing of certain specific mutations (e.g. at -12, -26(12) , -52 to
-50, and -36 reported here) when the URS is uncoupled from
the DRS is difficult to explain solely on the basis of two promoters. lacZ fusions to either the StuI or XmaIII
sites located approximately 225 and 92 bp, respectively, upstream of
the start site of transcription, result in only background levels of lacZ expression under various conditions of growth. For
example, in the presence of high oxygen the wild type level of LacZ is
163 units of
-galactosidase/mg of protein, whereas the XmaIII fusion yields
4 units, and the StuI
fusion yields
9 units. Similarly, under dark/Me
SO
conditions the wild type yields
1533 units and the XmaIII
and StuI fusions yield
8 and 11 units, respectively.
These low levels of
-galactosidase from the upstream fusions are
similar to those observed when lacZ is positioned immediately
downstream of an
cartridge without any obvious promoter
sequences positioned in between. Similar experiments conducted with
cells growing under low light intensities have given similar results.
It would be necessary to assume that changes in the DRS could affect
the upstream promoter, leaving us back where we started. Finally, the
exceptionally low levels of residual puc operon expression as
well as the absence of any increased expression following the removal
of O
in the
d mutation lying entirely within the DRS
supports the existence of a single promoter within the DRS. Therefore,
without evidence to the contrary we will continue with the working
hypothesis that the interaction of the URS with the DRS is through the
mediation of additional trans-acting factors. Our laboratory
has defined several potential factors and, as described earlier, an FNR
homologue.
Where is the promoter within the DRS located?
Previously (12, 14) we proposed that the regulatory
region of the puc operon possessed the properties of both
- and
-type
promoters(27) . In either instance RNA polymerase can cover DNA
coordinates between -50 and +20. In light of the effects of
the
d deletion on puc operon expression we suggest that
the most upstream portion of the promoter has been removed. This
interpretation is complicated by the fact that the
d deletion has
resulted in the removal of less than two helical turns of the DNA,
placing the putative activator binding region out of phase with the
promoter. However, the
c mutation that removes the putative
activator binding sequence does not diminish puc operon
expression to the extent of the
d mutation, and more importantly,
the
c mutation leaves ± 0
control largely
intact. Therefore, we propose that the promoter for the puc operon overlaps both the distal and proximal regions of dyad
symmetry. This is supported by recent work by Gomelsky and Kaplan. (
)A similar although not identical case has been observed in
the regulatory region of the bchCXYZ operon in R.
capsulatus(28) .
Comparison of the 90-bp puc upstream DNA of R. sphaeroides with that of R. capsulatus(25, 26) reveals a high degree of sequence homology from -84 to -66 (putative activator binding regions(s)) and -52 to -28 (distal dyad symmetry and its immediate 7-bp downstream DNA) as well as from -9 to -1. Only one sequence of dyad symmetry was found in the puc upstream DNA from R. capsulatus(25, 26) .
Penfold and Pemberton (29) recently identified a gene, ppsR, which alone is
sufficient for photopigment suppression in R. sphaeroides. The ppsR gene encodes a protein with a DNA-binding domain within
the COOH-terminal region. These authors suggested that the PpsR protein
recognizes the motif TGT-N-ACA, found to reside within the
immediate cis-acting sequences of some but not all
photopigment genes. We proposed (12) that these same sequences
within the DRS of the puc operon are involved in factor
binding(17) . In a more direct approach to this hypothesis
Gomelsky and Kaplan
have determined that the ppsR gene product interacts with these regions of dyad symmetry and
that point mutations within these sequences can alter PpsR-mediated
repression of puc operon expression.
We now possess genetic
evidence that the region of dyad symmetry possessing the
TGT-N-ACA motif has repressor binding activity.
Because these same regions of dyad symmetry occupy the region
-10 to -52, this sequence may also contain the promoter for
the puc operon. The net effect of mutations within these
sequences could simultaneously reflect alterations in both repressor
binding and promoter strength. For example, point mutations having
increased puc operon expression in the presence of O
may reflect diminished repressor binding without any effect upon
the puc promoter. However, we cannot rule out the possibility
that other mutations could lead to increased promoter strength without
affecting repressor binding. Intermediate alternations between these
extremes are more than likely. It is also likely that each region of
dyad symmetry is capable of binding repressor, since mutations mapping
to both regions lead to increased puc operon expression in the
presence of O
.
If we assume that under anaerobic conditions repressor binding to the region(s) of dyad symmetry is no longer involved in regulating expression of the puc operon, then the mutations at -12, -36, and -52 to -50 may reflect changes in promoter accessibility, the former two directly representing changes in promoter sequence and -52 to -50 indirectly through their effect on the -36 region by virtue of a change in DNA structure. Since these changes are only observed in the presence of the URS, we conclude that the presence of the URS is important for enhanced accessibility of RNA polymerase to the puc operon promoter.
There are two mutations, at -11 and
-37 to -35, which in addition to displaying increased puc operon expression in the presence of O do so
whether or not the URS is coupled to the DRS. Each of these changes
occurs in the proximal arm of both the distal and proximal regions of
dyad symmetry. Several interpretations, which are not mutually
exclusive, are possible. These mutations might signify asymmetric
binding of the repressor to the region(s) of dyad symmetry. Or these
sequences may lie within and therefore define the puc operon
promoter.
At this point it is worth noting that mutations in the URS
sequence located between -197 and -190 led to a 7-fold
increase in puc operon expression in the presence of
O. (
)This observation is most readily explained
if direct interactions, mediated by protein factors, between the URS
and DRS can occur. Interactions between the URS and the DRS are further
strengthened by the results obtained with the duplication of the region
from -34 to -23. In the absence of the URS, RNA polymerase
interactions would be destablized and activity would be low, as seen.
However, in the presence of the URS and other protein factors proper
alignment of those regions might be favored. Any explanations involving
transcription initiation from sequences within the URS would still
require additional mechanisms for such changes in the URS resulting in
silencing of repressor binding within the unaffected DRS.
It is obvious that much remains to be learned regarding the cis-acting regulatory sequences involved in puc operon expression. If we are correct in our assumptions that the regions of dyad symmetry within the DRS serve both to bind repressor (and perhaps other regulatory effectors) and RNA polymerase, then the mutational effects observed here represent the outcome of a subtle interplay between these disparate activities. Therefore, it may be possible to view each of these unique roles through the use of specific genetic backgrounds. For example, in a PpsR minus background it may be possible to undertake an investigation of the puc operon promoter, all else being equal.