Download Structure-Function Relationship and Regulation of Two Bacillus

J. Mol. Microbiol. Biotechnol. (2002) 4(3): 323–329.
JMMB Symposium
Structure-Function Relationship and Regulation
of Two Bacillus subtilis DNA-Binding Proteins,
HBsu and AbrB
Wolfgang Klein1,2, and Mohamed A. Marahiel 1*
1
Philipps Universität Marburg, Biochemie - FB Chemie,
Hans-Meerwein-Strasse, D-35032 Marburg, Germany
2
Institut für Pharmazeutische Biologie, Rheinische
Friedrich-Wilhelms Universität, Nussallee 6, D-53115
Bonn, Germany
Abstract
Microorganisms use a number of small basic proteins for organization and compaction of their DNA.
By their interaction with the genome, these proteins
do have a profound effect on gene expression,
growth behavior, and viability. It has to be distinguished between indirect effects as a consequence
of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase
action as represented by the histone-like proteins,
and direct effects by specific binding of proteins
to defined DNA segments predominantly located
around promoter sequences. This latter class is
represented by the transition-state regulators that
are involved in integrating various global stimuli
and orchestrating expression of the genes under
their regulation for a better adaptation to changes
in growth rate. In this article we will focus on two
different but abundant DNA binding proteins of
the gram-positive model organism Bacillus subtilis,
the histone-like HBsu as a member of the unspecific
and the transition state regulator AbrB as a member
of specific classes of DNA binding proteins.
The HBsu Protein of Bacillus subtilis is a
Member of the Histone-like Protein Superfamily
More than 30 members of the family of histone-like
proteins, all with a size of about 90 amino acids and an
overall basic net charge, have been identified so far in
organisms of virtually every branch of the eubacterial
kingdom, archaea, cyanobacteria, plant chloroplasts,
and bacteriophages (Oberto et al., 1994). The B. subtilis
genome encodes for one histone-like protein by the hbs
gene (Kunst et al., 1997). This correlates with findings
that the hbs gene is essential, and attempts to construct
a knock out mutation have failed so far (Micka and
Marahiel, 1992). The HBsu protein shows a high level
*For correspondence. Email marahiel@ chemie.uni-marburg.de;
Tel. +49 +6421 -2825722; Fax. +49 +6421 -2822191.
# 2002 Horizon Scientific Press
of similarity to the E. coli heterodimeric HU protein, the
so far best studied histone-like protein. It shows 57%
and 51% identical amino acids with the HU-subunits
HU-2 and HU-1, respectively. From the occurrence of a
single gene in B. subtilis genome it is concluded that
HBsu acts as a homodimer (see Figure 1A), and our
data on the DNA-binding activity of HBsu mutant proteins support this idea. When subsaturating protein
concentrations of one protein were used, they exhibited
full DNA binding activity by the addition of subsaturating amounts of another mutant variant (Köhler and
Marahiel, 1998), indicating the formation of a functional
complex of both mutant proteins. The construction of a
HBsu-GFP fusion protein allowed us to visualize for
the first time the cellular localization of this protein as
in vitro analysis has shown its ability to bind DNA. Using
fluorescence microscopy, HBsu-GFP fluorescence
was observed inside the cell at exactly the same location as the DAPI fluorescence that is localizing the
nucleoid. Furthermore, additional expression of wildtype HBsu caused the formation of a more condensed
nucleoid (Köhler and Marahiel, 1997). These results
and investigations using HBsu proteins with reduced
DNA binding activity emphasize its principal role in DNA
packaging in B. subtilis.
The archetype of histone-like proteins is H-NS of
Escherichia coli that has been identified several times
independently according to the researchers focus on
functions like in vitro transcription of phage DNA, phage
transposition, and lifestyle (for a review, see Nash,
1996). In vivo localization data of this heterodimeric
protein yielded controversial results by showing correlation with metabolically active DNA in immunocytochemical studies and even distribution throughout
the entire nucleoid using fluorescein labeled HU, a
H-NS homologous protein (Shellman and Pettijohn,
1991). A recent model of nucleoid organization in E. coli
suggested that HU and H-NS are located in different
domains where an internal domain, thought to contain
rarely transcribed DNA, is associated with H-NS while
frequently transcribed sequences complexed with HU
protein form the coralline appearing outer sphere of
the nucleoid (McGovern et al., 1994). Although these
two histone-like proteins appear to play special roles in
cellular physiology, E. coli strains lacking either HU or
H-NS are viable whereas multiple mutations severely
impair cellular viability (Yasuzawa et al., 1992).
Structural Features of HBsu
An understanding of the molecular structure of histonelike proteins is mainly based on X-ray crystallographic
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324 Klein and Marahiel
analysis of the homodimeric HBst protein from
B. sterothermophilus (Tanaka et al., 1984); see Figure
1A). As HBsu of the mesophilic B. subtilis and HBst
display 87% identity and 94% similarity, a nearly
identical 3D-structure for both can be assumed. The
amino terminal half of the HBst protein is composed of
two a-helices connected by a turn, followed by three
b-sheets. Whereas the first sheet is involved in forming
the body of the protein structure, the latter two form a
b-ribbon extension called the ‘‘arm’’ that was shown to
be involved in DNA binding (Köhler and Marahiel, 1998;
see Figure 1A). The interface of the dimer is largely
dependent upon interactions between several hydrophobic residues scattered along the primary sequence,
an attribute that is conserved in all members of this
family of proteins.
DNA-Binding by HBsu
DNA binding by HBsu is independent of cofactors or
additional proteins. It has been shown to bind DNA
unspecifically but with a preference for curved fragments (Köhler and Marahiel, 1998). Furthermore, HBsu
enables b-recombinase-mediated recombination by
stabilizing a special DNA structure (Alonso et al.,
1995). Even though experimental data are lacking, the
preference for curved DNA could be an indication that
HBsu might perturb the overall structure of duplex DNA,
a fact that has been seen for the E. coli HU protein
(Hodges-Garcia et al., 1989).
In a model based on the HBst structure, each
monomer of the homodimer contributes two DNA
binding surfaces – the b-ribbon ‘‘arm’’ and a segment
on the flank of the body - both of which contact the minor
grove of the DNA. Based on a model that has been
deduced from DNA-protein photo-crosslinking experiments with a similar DNA-binding protein (Lee et al.,
1992; Yang and Nash, 1994), we have performed the
first site-directed mutational analysis of a histone-like
protein. Our results strengthened the significance of
several basic residues in DNA binding as the mutated
proteins showed a dramatically reduced DNA binding
activity (see Figure 1C). Furthermore, this in vitro activity is corroborated by in vivo data showing a reduced
growth rate, a reduced sporulation efficiency, and a less
condensed nucleoid for strains carrying only a mutant
hbs allel (Köhler and Marahiel, 1998).
Regulation of hbs Expression
There are only few data on the regulation of histone-like
proteins that may explain the abundance of HU protein
in E. coli in response to different growth conditions.
Values of 2 to 5 ng of HU per mg total protein might
represent an average that varies little, if at all, in the life
cycle (Bonnefoy et al., 1994). This might reflect the
operation of an autoregulatory circuit, yielding an
appropriate amount of HU protein and resulting in a
suitably condensed nucleoid (Kohno et al., 1990). Our
own data on the regulation of the B. subtilis hbs gene
are based on two lines of evidence: two promoter
structures in front of the hbs gene were mapped. P1 is
181 nucleotides upstream of the translation start point,
shows similarities to sA dependent promoters, and the
mRNA derived from this promoter was detected mainly
in exponentially growing, non-sporulating cells. P2
localizes only 21 basepairs upstream of the ATG
codon, and its sH promoter similarity is corroborated
by determining this type of mRNA exclusively in
sporulating cells (Micka et al., 1991). Furthermore,
different promoter fragments have been cloned in front
of the b-galactosidase gene and integrated as single
copy into the chromosome for monitoring hbs::lacZ
expression (Köhler and Marahiel, 1997). The P1 and P2
comprising fragments triggered a high b-galactosidase
activity with a slight maximum around one hour before
the transition into stationary phase. Studies on different
P1 promoter fragments revealed two facts: a medium
level of hbs::lacZ expression during exponential growth
but low expression after the transition phase. Deletion
of a short DNA segment located between both promoter
structures, P1 and P2, selectively reduced expression
by promoter1 under all conditions tested. We therefore
attributed a P1-specific enhancer function to this DNA
segment. In addition, P1 seems to be the target of the
HBsu autoregulatory loop as conditional overexpression of wildtype HBsu selectively reduced hbs:lacZ
expression directed by P1 but not by P2. Fragments of
promoter2 showed only a low activity that is about 12
fold induced around one hour after entry into the
sporulation phase. This induction is dependent on
sporulation promoting growth conditions and strictly
dependent on sH as tested in spo0H mutants (Köhler
and Marahiel, 1997). Based on these data, we propose
that HBsu is regulated in exponentially growing cells by
P1 and an autoregulatory loop. After the transition to
stationary phase either expression is promoted by the
sH dependent P2 if sporulation is initiated, or expression of hbs is reduced under non-sporulating conditions
and HBsu might be in part replaced by proteins like the
minor small, acid-soluble spore proteins (Ross and
Setlow, 2000).
AbrB of Bacillus subtilis is the Major
Transition State Regulator
At the end of exponential growth in a bacterial culture
when conditions for growth become sub-optimal, many
genes for alternative catabolic and anabolic pathways
to produce substances such as antibiotics, toxins, and
polymer degrading enzymes become derepressed.
Therefore, the transition state between exponential
growth and stationary phase can be looked at as a
crossroad where cells still express some growthrelated functions but begin to express new gene
products that are necessary for survival in a nutrientdeprived, hostile environment. In Bacillus spp, typical
functions of the transition state encompass
the development of genetic competence for the uptake
of DNA, production of antibiotics and extracellular
enzymes, and the synthesis of flagella. Genes encoding such functions are regulated, at least in part, by
small, global negative transcriptional factors that have
been termed ‘‘transition state regulators’’ as they si-
HBsu and AbrB Proteins of Bacillus subtilis 325
Figure 1. Structural and functional features of HBsu as a member of the histone-like protein family. A) The three-dimensional structure of the
homodimer HBst protein of B. stearothermophilus was used as a model for site-directed mutagenesis in HBsu of B. subtilis. The basic residues
selected for mutagenesis are shown with their side chains and labeled with single-letter amino acid code in both proteins. N- and C-termini are
indicated by n and c, respectively, numbers refer to the two subunits. Modelling of the structure was done according to Konradi et al., 1996. B)
Comparison of the amino acid sequences of HBsu and HBst which are 86% identical. Mutationally altered amino acid residues are indicated above
the sequence, secondary structure elements of HBst are shown schematically below the sequences. C) Gel retardation analysis of HBsu and mutant
proteins with curved and non-curved DNA.
326 Klein and Marahiel
lence the expression of these functions at inappropriate times (Strauch, 1993). None of the known mutations in these regulators leads to significant
sporulation defects, but they are involved in coordinating and transmitting signals received by the sporulation-sensing network and orchestrating the transition
state functions. One of these regulators is the product
of the abrB gene that controls several transition state
genes, some of which are necessary for sporulation
(Perego et al., 1988). In this manner, AbrB coordinates
the sporulation response with other transition state
processes. Furthermore, AbrB is implicated in the
regulation of other transition state regulators such as
Hpr (originally identified by mutations causing overexpression of extracellular proteases (Perego and
Hoch, 1988)) and Sin (inhibitor of sporulation and
protease production when overexpressed (Gaur et al.,
1988; see Figure 2)). AbrB is looked at as the
central transition state regulator in Bacillus subtilis.
The abrB gene was isolated as pseudorevertants in
a spo0A mutant background (spo0A mutants show
impaired antibiotic production, lack of motility, inability
to aquire genetic competence and to initiate sporulation) by restoring antibiotic production and motility but
not sporulation (Guespin-Michel, 1971). Cloning of this
locus revealed a single gene encoding for a 94 amino
acid protein (10.5 kDa), and the pseudorevertants
isolated resulted in loss of function (Perego et al.,
1988) with one class lowering the expression of abrB,
while the other class impaired the reading frame (Zuber
and Losick, 1987). The abrB gene is transcribed by two
promoters, whose physiological significance is not yet
fully understood.
DNA Binding of AbrB
Attempts to define common binding sites for the
genes regulated by AbrB have failed so far. The
location of AbrB-binding sites relative to the promoters shows some flexibility but suggests that AbrB
might interfere or interact with the RNA polymerase
(Strauch, 1995a). Footprinting analysis showed variation in size from target to target, with protected
regions ranging from 30 to 120 base pairs (Strauch,
1995b). However, from these sequences no common
motif can be deduced as an AbrB consensus
sequence. Using an in vitro selection of random oligo
nucleotides, seemingly optimal AbrB binding sites
corresponding to a regularly spaced motif were
selected (Xu and Strauch, 1996). This optimally
spaced motif, however, is rare within the B. subtilis
genome and does not correlate with the footprinting
results. Therefore it is not sufficient to explain the
selectivity of the AbrB-DNA-interaction. The mapped
sequences show a correlation to AT-rich and bent
DNA regions, but bending alone is not sufficient for
promoting AbrB binding as shown by mobility shift
assays using bent DNA (Klein, unpublished data).
Therefore, it has been hypothesized that AbrB may
actually recognize a type of DNA-three-dimensional
structure that can be assumed by a finite subset of
differing base sequences (Xu et al., 1996).
The 10.5 kDa AbrB protein has been shown to be a
hexamer in solution, and that this hexameric state is
required for its DNA-binding activity (Strauch et al.,
1989). A sequence with slight similarity to the helix-turnhelix motif of DNA-binding proteins that was identified
within the AbrB sequence was investigated by mutational analysis and failed to prove this relationship
(Fürbaß and Marahiel, 1991). There is no evidence for
the presence of any other known DNA-binding motif
within the AbrB protein. Therefore at present no
explanation can be provided for the specific and
co-operative binding behavior of AbrB (Fürbaß et al.,
1991; Robertson et al., 1989).
To understand the structural basis of AbrB specific
DNA-interaction, several attempts to unravel the three
dimensional structure of this proteins have been
undertaken. These studies were designed to define
the residues involved in DNA binding and to shed light
on the oligomerization behavior. Full-length recombinant AbrB protein from B. subtilis was purified to
homogeneity but failed to crystallize. Recently the
structure of the AbrB N-terminal DNA-binding domain
was solved by NMR. It was described as a loopedhinged helix fold where two conserved arginine residues required for DNA interaction are located within an
a-helix flanked by a double stranded looped-hinge
region (Vaughn et al., 2000). This N-terminal domain
was shown to retain full sequence specificity and to
form dimers that are necessary for DNA interaction, but
it failed to show the hexameric state that has been
described for the full-length protein. NMR measurements showed that the residues of the hinge-loop
region undergo significant concerted motion upon the
protein’s interaction with DNA, affecting the disposition
of the DNA-binding helix (Zuber, 2000). According to
the recent model of gene regulation by AbrB, the
ordered structure of AbrB-DNA complexes varies
between different target genes, suggesting changes
within AbrB’s tertiary and quarternary structure to
conform to the site of interaction offered by the DNA
target. This might be accomplished by the multimeric
state of AbrB as determined by the specific target
(Vaughn et al., 2000).
The validity of the proposed model can only be
tested on the structure of full-length AbrB bound to
different DNA-target sites. Such an investigation will
also reveal all the amino acid residues mediating direct
interaction with the DNA, and those needed for the
multimeric organization. As a first step towards structural studies on full length AbrB protein, we isolated
the abrB gene from the thermophilic Bacillus stearothermophilus (76% identity to the mesophilic B. subtilis
protein). DNA-binding activity and oligomerization
behavior of the recombinant protein were studied and
displayed a similar behavior as shown for the mesophilic
AbrB su. Initial crystallization attempts yielded small
crystals that show diffraction at 4 Å (Klein et al., 2000).
Regulation of Gene Expression by AbrB
AbrB has been characterized to act in three different
ways to regulate post-exponential expression of genes
HBsu and AbrB Proteins of Bacillus subtilis 327
Figure 2. AbrB regulation of transition state gene expression. +, positive regulation; ! , negative regulation. The formation of the phosphorylated,
active form of Spo0A occurs at the end of exponential growth by a phosphorelay which is summarized here. t 0 means time zero according to the
sporulation pathway. The components of this regulatory circuit and their cellular functions are summarized.
which can be classified according to their cellular
function as summarized in Figure 2. It is the sole
repressor for genes like spo0E and tycA as abrB
mutations cause constitutive expression of these genes
(Marahiel et al., 1987; Perego and Hoch, 1991). For
spoVG and aprE, AbrB is termed a ‘preventer’: while
abrB mutations restore their expression in Spo0
mutants, the absence of AbrB does not cause constitutive expression, indicating additional temporal control
acting in concert with AbrB (Ferrari et al., 1988). In a
third manner AbrB regulates expression as an activator.
Genetic data indicate that AbrB positively affects
expression of the transition state regulator Hpr (Strauch
and Hoch, 1993), factors of the competence pathway
(Dubnau, 1991), and enzymes involved in histidine
utilization (Fisher et al., 1994). Whereas in the case of
repression the binding of AbrB to DNA segments around
the promoter region has been verified, this has not yet
been shown clearly for positively regulated genes. By
the combination of several levels of macromolecular
assembly with the flexibility of the looped-hinge helix
DNA recognition fold to alter its orientation, AbrB can
serve as specific regulatory protein for diverse promoter
elements. This structure-function model sets AbrB as
an archetype for a new class of regulatory proteins
(Zuber, 2000).
To date, abrB expression is known to be controlled
by both negative autoregulation and repression by
phoshorylated Spo0A protein (Perego et al., 1988). It
is believed that the autoregulatory loop ensures
an intracellular concentration of AbrB during exponential growth high enough to silence transition state
functions and sensitive enough to changes in transcription and/or protein activity. The cooperative DNAbinding activity of AbrB (see above) could magnify the
effects of slight decreases in the AbrB levels on its
328 Klein and Marahiel
target genes. Phosphorylated Spo0A protein, being
the primary sporulation signal as it is phosphorylated
by a regulatory phosphorelay cascade in response to
nutrient deprivation, seems to be the sole factor
responsible for abrB repression (Strauch et al.,
1990). This sporulation initiating signal cascade
represses transcription of the abrB gene and causes
the AbrB level to drop below its threshold, thereby
releasing the repression of transition state functions.
This genetic model is supported by data showing that
the cellular amount of AbrB is indeed growth phase
dependent (O’Reilly and Devine, 1997). Both abrB
mRNA and AbrB protein accumulate in the early
exponential growth phase, but whereas the transcript
level sharply decreases in the mid-exponential phase,
the AbrB protein is gradually decreasing along the
exponential growth with an abrupt reduction at the
onset of stationary phase. Furthermore, analysis of
abrB (partially deletion) and spo0A mutant strains
confirmed the abrB regulation model on the level of
mRNA and protein quantification (O’Reilly and Devine,
1997). Even though not related on the amino acid
level, the pattern of abrB expression, its size, and
overall net charge are similar to that of Fis from E. coli.
But both proteins bind to highly degenerate consensus
sequences with a bias for A and T residues, both are
negatively autoregulated, and both regulate expression of a variety of genes of diverse functions.
Interestingly, so far no Fis homolog has been found
in gram-positive bacteria, and AbrB seems to be a
characteristic of the genus Bacillus. This has been
taken as an indication that these two proteins evolved
independently but have similar cellular functions
(O’Reilly and Devine, 1997).
Acknowledgements
We would like to thank all the members in our lab that have contributed
to the research in these topics as well as all our colleagues that have
sheared ideas and results. We apologize to all those whose work was
not cited appropriately due to limitations in article size. Our research
was supported by grants from the DFG.
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