(Received for publication, September 18, 1995; and in revised form, November 10, 1995)
From the
The Bacillus subtilis global regulator AbrB is a
DNA-binding protein composed of six identical monomers of 96 amino
acids that shows specificity to the promoter regions of its target
genes including its own. We have sequenced thirteen previously
uncharacterized abrB mutations. Four mutant AbrB proteins were
purified, and their DNA-binding properties and multimeric structures
were examined. AbrB23 (R25S) had no appreciable DNA binding activity
but retained a hexameric structure, indicating that Arg25 is important
in DNA interactions. Three other mutant proteins, AbrB1 (C56Y), AbrB19
(Gln
termination codon), and AbrB100 (L69P), showed
decreased DNA binding and altered multimeric interactions. Analysis of
the expression and AbrB binding affinities of mutant abrB promoters demonstrated that a consensus -35 region is
incompatible with proper autoregulation of the abrB gene.
AbrB is a DNA-binding transcriptional regulator of numerous genes that commence expression at the end of vegetative growth and the onset of stationary phase and sporulation(1, 2) . Its primary function is to prevent inappropriate expression of nutrient limitation-induced functions at times when they are not needed. The regulatory role of AbrB is not limited to events associated with growth cessation because it also modulates expression of some genes during slow exponential growth in suboptimal environments. In at least two cases, histidine utilization (3) and ribose transport(4) , this involves antagonism of the catabolite repression response caused by slowly metabolizable sugars.
AbrB does
not share significant amino acid homology with other known classes of
DNA-binding proteins(5, 6) . It is a hexamer of
identical small subunits (96 amino acids), and it appears that the
integrity of the hexameric structure is critical for DNA
binding(7) . DNaseI footprinting on over 20 chromosomal targets
has revealed defined protection regions ranging in size from 24 to over
100 contiguous base
pairs(3, 4, 8, 9, 10, 11) . ()Examination of these regions has failed to identify a
simple DNA consensus sequence that can be unequivocally assigned as a
recognition determinant(11) . It is believed that AbrB
recognizes a specific three-dimensional DNA structure that can be
assumed by a finite subset of differing base
sequences(1, 8, 11, 12) . Using in vitro selection of random oligonucleotides, we have
isolated seemingly optimal AbrB-binding sites that possess two to four
occurrences of a 5-bp (
)motif (TGGNA) separated by defined
spacings(12) . However, multiple regularly spaced examples of
this motif are rare in chromosomal binding sites. It is thought that
such optimal sites are infrequently used in vivo because they
are incompatible with promoter structures and with the mechanism
responsible for rapid relief of repressive effects due to AbrB-DNA
interactions.
Mutations in the promoter and coding regions of abrB have been isolated, and a few of them have been sequenced and characterized (7, 13, 14, 15, 16, 17, 18, 19) . In this communication, we report the sequence of additional abrB mutations and the functional analysis of mutant AbrB proteins and promoters. Three amino acid residues critical for either DNA binding or multimeric associations were identified. The differential biochemical properties of four mutant proteins suggests that the AbrB monomer might be divided into separable DNA-binding and multimerization domains.
Three mutations in the -35 region of the major abrB promoter (13) were analyzed with respect to in vivo promoter strength and their being subject to
autoregulation(7) . Our results resolve a previously noted (13) paradox: one class of mutations producing an AbrB
phenotype actually results in a perfect consensus to the -35
region sequence utilized by
RNA polymerase. We
demonstrate that in the absence of regulation, this promoter is indeed
more actively transcribed, but that the mutation responsible
concomitantly strengthens AbrB binding affinity, increasing the degree
of vegetative autoregulation in vivo, thus accounting for the
observed AbrB phenotype. Significantly, this mutation produces a
sequence that more closely resembles an optimal binding site selected in vitro. A promoter down mutation that diminishes AbrB
binding affinity shows less similarity to the optimal in vitro site. Both of these binding site/promoter mutations affect AbrB
interaction only with the higher affinity portion (-14 to
-43) of the total binding region (-14 to -125).
The electrophoretic
mobilities (R) of the proteins were determined and
100 log (R
1000) was plotted versus the percentage of gel concentration for each. Lines were fitted
using linear regression. The slopes (K
) were
compared with a standard curve obtained by plotting
-K
for each standard protein
(
-lactalbumin, carbonic anhydrase, chicken egg albumin, and bovine
serum albumin) versus their molecular weights.
Figure 1: Location and nature of abrB mutations. The numbering system of the nucleotide sequence is relative to the P2 transcription start site. -35, -10, and +1 portions of the promoter are underlined. @, amino acid sequence caused by the abrB20 mutation is given in parenthesis. AbrB3, 4, 6, 15, and 21 were sequenced previously. AbrB100 is a PCR-induced mutation that was generated in this study. ter, stop codon.
We feel that the molecular weight values obtained from our native polyacrylamide gel determinations underestimate the true sizes of the proteins. A previous study (7) using gel filtration chromatography indicated that wild-type AbrB was a hexamer with a molecular weight of 63,000. The discrepancy between the two types of determinations is probably related to the electric charge of the proteins.
Figure 2: Gel retardation assay of binding of mutant AbrB proteins to randomized oligonucleotides. Lane a, no protein; lanes b and c, wild-type AbrB; lanes d-f, AbrB1; lanes g-i, AbrB19; lanes j-l, AbrB23; lanes m-o, AbrB100. Protein concentrations are: 5 (lanes d, g, j, and m), 2.5 (lanes b, e, h, k, and n), and 1 µM (lanes c, f, i, l, and o). unb, unbound DNA.
We did
not undertake an analysis of the oligonucleotide sequences bound by the
mutant proteins, so it was not known if they represented the same
classes as those bound by wild type or if the binding activities of the
mutant proteins were also altered in specificity. To examine if the
altered DNA binding properties of the mutant proteins still retained
specificity characteristics of the wild type, we performed DNaseI
footprinting experiments on three different substrates. No protection
by AbrB1 or AbrB23 was seen using the spo0E site, but AbrB100
was observed to protect the same region as wild type, although much
higher protein concentrations were required (Fig. 3). Only
partial protections of the spo0E site were observed using the
higher concentrations of AbrB19 (Fig. 3). The BS16 and C34 sites
had been selected in vitro from pools of random sequence
oligonucleotides (12) and gave 25- and-45 bp DNaseI footprint
regions using wild-type AbrB. ()AbrB23 did not bind to these
sites, whereas AbrB1, AbrB19, and AbrB100 did show partial binding but
only at higher protein concentrations (data not shown).
Figure 3: DNaseI footprints of AbrB proteins on the spo0E binding site. Protein concentrations are 20 (lanes a), 4 (lanes b), and 2 µM (lanes c). Lanes R and Y are Maxam-Gilbert purine and pyrimidine sequencing reactions, respectively. The DNA target fragment is described under ``Experimental Procedures'' and was obtained from a clone of the 38-bp portion of the spo0E gene that corresponds to the AbrB-binding site. The sequence of this site is AATATGTTTACAAATAAAGTATAATCTGTAATAATGCA. It can be read from the sequencing ladder adjacent to the AbrB protection regions by using its complement and reading from bottom to top.
Figure 4:
In vivo expression from the abrB8 promoter. A, -galactosidase assay of
amyE::
(abrB8-lacZ) fusion in different
genetic backgrounds.
, wild type;
, abrB
;
, spo0A
;
, spo0A
abrB
. B, comparison of expression levels of
amyE::
(abrB
-lacZ)
and
amyE::
(abrB8-lacZ) in spo0A
and spo0A
abrB8 backgrounds.
, spo0A
amyE::
(abrB
-lacZ);
, spo0A
amyE::
(abrB8-lacZ);
, spo0A
abrB8
amyE::
(abrB
-lacZ);
, spo0A
abrB8
amyE::
(abrB8-lacZ).
For each graph, t
on the abscissa indicates the end of exponential growth of the
cultures.
Final proof that the abrB8 mutation results in a stronger AbrB binding affinity was obtained by in vitro DNaseI footprinting assays (Fig. 5). AbrB was able to protect the -14 to -43 region of the abrB8 promoter at much lower protein concentrations than those needed to see protection of the corresponding region of the wild-type promoter. Binding to the upstream (weaker affinity) region from -44 to -125 was largely unaffected by abrB8. We note that the region in the vicinity of -81 of the abrB8 promoter is more susceptible than the wild-type promoter to DNaseI cleavage, both in the presence and the absence of bound AbrB. This relative hypersensitivity implies that the single base pair change of abrB8 at -29 can in some way affect the overall DNA structure as far away as -81. We do not know the nature of this alteration, but it does not appear to significantly affect overall AbrB binding to the upstream region.
Figure 5: AbrB binding to wild-type and mutant promoters. The triangles indicate decreasing AbrB concentrations (10, 5, 2, 1, 0.5, 0.2, 0.1, and 0.05 µM). The AbrB-binding regions are denoted by a bracket with a thick line indicating the more strongly bound portion. The numbering system is relative to the P2 start site.
We also performed footprinting experiments on the abrB11 and absB24 promoters (Fig. 5). The abrB11 mutation caused no visible change in AbrB binding extent or affinity. The absB24 mutation resulted in a complete loss of AbrB binding at -14 to -43, but the affinity of binding at -44 to -125 was not significantly altered, and, in fact, the DNaseI cleavage site at -81 was protected by AbrB.
We have sequenced a number of previously uncharacterized abrB mutations, including two, tolB24 and absB24, which are now proven to be abrB alleles. The majority of mutations located within the abrB coding region are highly disruptive (deletions, insertions, frameshifts, and nonsense). Only two different naturally occurring mutations that change a single amino acid residue were discovered. Perhaps many more point mutations changing single residues are phenotypically silent.
The
AbrB23 protein (R25S) retains a hexameric structure but has completely
lost specific DNA binding activity (Fig. 3). It is likely that
it is also devoid of nonspecific DNA binding activity (see Fig. 2). This indicates that Arg is probably a
critical residue involved in interaction of the hexamer with DNA, but
the exact nature of the defect caused by its substitution with serine
is unknown. Deducing the roles of the residues altered due to abrB1 (C56Y), abrB100 (L69P), and abrB19 (Q83ter) is
more problematical. In each case, the mutant proteins exhibit altered
subunit interactions, resulting in the predominant multimeric forms
being smaller than wild type (Table 1). As expected, all three
are also defective in DNA binding properties but not to as severe an
extent as AbrB23 ( Fig. 2and Fig. 3). However, these data
do not allow a definite conclusion that the sole roles of
Cys
, Leu
, or the carboxyl-terminal 14 amino
acids are in formation of the multimer; some or all may also directly
interact with DNA.
The C56Y, L69P, and Q83ter alterations do not
completely destroy multimerization. The major proportion of the mutant
proteins are in either trimer or tetramer forms with no evidence of
significant dissociation into monomers. Although Cys is
the sole cysteine residue in the monomer, it is unlikely that it
participates in disulfide bonds between subunits. The results of the
native gel determinations of molecular weights presented here indicate
that AbrB1 is a tetramer, but a previous determination using gel
filtration showed that a C56Y protein retained the hexameric
configuration(7) . We believe this discrepancy is related to
the different methods used and that the Cys
Tyr
change probably weakens subunit interactions with the effect being
apparent only under the conditions of the native gel method (e.g. migrating in an electrical field). Additionally, using
iodoacetamide and nonreducing gel methods (29, 30) ,
we have found no evidence that disulfide bonds exist in wild-type AbrB. (
)
Recently, within the context of the B. subtilis genome sequencing project, two genes encoding proteins with high
amino acid homology to AbrB have been
discovered(31, 32) . One encodes a 92-amino acid
protein whose first 50 residues show 74% identity to residues
3-52 of AbrB(31) . The other, designated spoVT, ()encodes a 178-amino acid protein whose first 51 residues
show 68% identity to AbrB residues 3-52. Although the DNA binding
properties of these proteins have not yet been reported, they both have
been shown to affect transcription of genes associated with stationary
phase and sporulation.
(
)Given that AbrB and
these two proteins are regulators of gene expression during a
differentiation process, it is tempting to speculate that the shared
homology over the first 50 or so amino acids represents the B.
subtilis equivalent of the eucaryotic
homeodomain(33, 34) . Functional similarities between
AbrB and homeodomain proteins have previously been noted(2) .
A high proportion of the total number of abrB mutations
examined occur in the promoter region. This probably reflects a bias in
the original methods used to select abrB mutations as partial
revertants of Spo0A phenotypes. It is now known that spo0A
abrB cells grow faster than spo0A
abrB
(3, 4) . Because some
promoter mutations could still express low levels of active AbrB (too
low, however, to give an AbrB
phenotype for the
characteristic used in the selection process), these might have grown
faster and been more noticeable in the selection. Supporting this
conclusion is the fact that for some AbrB phenotypes there is a
detectable difference between promoter and coding
mutations(3, 13, 19) .
The -35
region of the abrB promoter has one mismatch to the
consensus, but six identical independently isolated
mutations presented a paradox: they resulted in a perfect -35
consensus. We have solved this paradox by showing that although the
mutation to the perfect consensus does result in a stronger promoter,
it concomitantly results in stronger negative autoregulation due to an
increased affinity for AbrB binding in the region ( Fig. 4and Fig. 5).
Autoregulation of abrB had previously been
correlated with AbrB binding to the -14 to -125 sequences
of the promoter(7, 8) . Within this large region, the
area from -14 to -43 appeared to have higher binding
affinity than the rest, as judged by DNaseI footprinting
assays(8) . Our in vivo expression studies and in
vitro footprinting assays of AbrB binding to the mutant abrB8 and absB24 promoters provide evidence that binding at the
-14 to -43 sequences is the major component responsible for
autoregulation. Although the abrB8 mutation increases the
binding affinity of AbrB at -14 to -43, it does not have an
effect on the binding affinity to the upstream region (Fig. 5).
As discussed above, abrB8 is more strongly autoregulated than
wild type. However, analysis of the absB24-lacZ fusion
revealed only a 2-4-fold difference between wild-type and spo0A abrB backgrounds during vegetative growth (data not
shown), suggesting that only autoregulation had been removed because
this magnitude of difference is identical to that seen for the native
promoter in wild-type versus spo0AabrB
cells(7) . (
)As seen
in Fig. 5, AbrB can still bind to the -44 to -125
sequences of the absB24 promoter but does not bind at
-14 to -43. Furthermore, AbrB binding affinity to the
upstream (-44 to -125) region is independent of the
presence (abrB8) or the absence (absB24) of AbrB
bound at -14 to -43, indicating that no cooperativity
exists between the regions. The role of the upstream binding region is
unknown, but perhaps it functions to increase the localized
concentration of AbrB immediately available for ``rebinding''
if dissociation of the adjacent AbrB (-14 to -43) complex
occurred.
In a previous study(12) , we had selected optimal in vitro AbrB-binding sites that were characterized by
regularly spaced repeated examples of a 5-bp motif (TGGNA; TNCCA on the
opposite strand) and had shown that they could confer AbrB-mediated
regulation in vivo. Nevertheless, examination of native
AbrB-binding regions did not reveal a significant occurrence of these
regularly spaced motifs. This seeming incongruity was explained by the
hypothesis that optimal AbrB binding sites were probably incompatible
with the function of most promoters and with the mechanism that leads
to the rapid release of AbrB-mediated repression at the onset of
stationary phase. Our footprinting results (Fig. 5) on the
wild-type and mutant promoters support this hypothesis. The relative
orientation of the in vitro selected motifs did not appear to
be a critical factor for AbrB binding, and one class had examples of
the type TNCCA-5bp-TGGNA. From -34 to -20 of the abrB promoter is a 15-bp sequence with partial homology to this
arrangement of dual motifs. The downstream motif (-24 to
-20) is a perfect match to TGGNA, but the aligned upstream motif
only matches two of the defined positions in TNCCA (Fig. 6).
However, the abrB8 mutation, which increases AbrB binding
affinity (Fig. 5), results in three matches in the upstream
motif; the absB24 mutation shows only one match and lowers
AbrB binding affinity; the abrB11 mutation only changes the
variable position (retaining the two wild-type matches) and has a
binding affinity comparable with the unaltered promoter. Additionally,
dual motifs found in the in vitro selected binding sites were
usually spaced 5 bp apart(12) . That this spacing (which places
the motifs on the same face of the helix) is important for AbrB binding
was evidenced by an in vitro generated variant of the
-43 to -14 abrB region that possessed a deletion
of two bases (Ts at -27 and -28) between the wild-type
TGACG and TGGAA motifs and did not bind AbrB in DNaseI and methylation
protection assays. ()
Figure 6: Comparison of wild-type and mutant promoter sequence to an in vitro derived consensus sequence for AbrB binding. The AbrB-binding region is shown as a bracket with a thicker line indicating the more strongly bound portion. The -35 and -10 promoter elements are shown in boldface type.