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Previous Article | Next Article 
Journal of Bacteriology, September 1999, p. 5384-5388, Vol. 181, No. 17
0021-9193/99/$04.00+0
Role of the Sporulation Protein BofA in Regulating
Activation of the Bacillus subtilis Developmental
Transcription Factor 
K
Orna
Resnekov*
Section on Microbial Genetics, Laboratory of
Molecular Genetics, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland
20892-2785
Received 1 March 1999/Accepted 21 June 1999
 |
ABSTRACT |
During sporulation, the Bacillus subtilis transcription
factor 
K is activated by regulated proteolytic
processing. I have used a system that facilitates the analysis of the
contributions of a modified form of the processing enzyme, SpoIVFB-GFP,
and the regulatory proteins BofA and SpoIVFA to the conversion of
pro-K to K. The results show that in the
presence of BofA, SpoIVFA levels increase by greater than 20-fold,
SpoIVFA is substantially stabilized, and pro-K
processing is inhibited. In addition, enhanced accumulation of the
SpoIVFA protein in the absence of BofA (achieved through the use of an
ftsH null mutation) substantially inhibits
pro-K processing. These results suggest that during
growth, increased accumulation of the SpoIVFA protein inhibits the
activity of SpoIVFB-GFP and regulates the activation of
K.
| |
INTRODUCTION |
During the process of sporulation in
the bacterium Bacillus subtilis, asymmetric positioning of a
septum partitions the developing cell into two compartments.
Subsequently, the smaller compartment (the forespore) is engulfed by
the larger compartment (the mother cell), to create a protoplast within
a cell. In the developing sporangium, transcription is controlled by a
cascade of four sporulation-specific transcription factors ( factors), whose activities are compartment specific and regulated
(6, 8, 15, 25). The mother cell transcription factors
E and K are activated by proteolytic
removal of their NH2 termini, 27 amino acids in the case of
E (19) and 20 amino acids in the case of
K (3, 14, 24). These regulated proteolytic
events are governed by signal transduction pathways that originate in
the forespore (11, 25). While the components and regulation
of the two pathways differ, both delay gene expression in the mother
cell until a signal has been received from the developing forespore and
both are thought to be important for coordinating the two-compartment programs of gene expression (25).
Following engulfment, processing of pro-K to
K takes place in the mother cell and requires the
protein SpoIVFB (3, 4, 16, 21, 22). SpoIVFB is the
processing enzyme or a required regulator of a presently
unidentified processing complex (16, 22) and, consistent
with its proposed function, contains a motif associated with
Zn2+-dependent endopeptidases (14a, 16). SpoIVFB
is inferred to be held inactive by the regulators BofA and SpoIVFA
(3, 4, 10, 22, 23). All three proteins are produced in the
mother cell (4, 10, 23) and are integral membrane proteins
(21, 26). SpoIVFA and SpoIVFB localize to the boundary
between the mother cell and the forespore (21) and are
encoded in the two-cistron spoIVF operon (4). The
localization pattern of BofA is unknown. Activation of SpoIVFB requires
the signal protein SpoIVB, which is produced in and presumably secreted
from the forespore (2, 7). In the absence of the signal
protein, processing of pro-K and
K-directed gene expression can be activated by mutations
in bofA or specialized mutations in spoIVFA
(bofB mutations); in these cases, processing occurs about
1 h earlier than in wild-type cells (3, 4, 23).
Vegetative B. subtilis cells engineered to induce two
proteins normally made only during sporulation, the proprotein
pro-K and a modified form of SpoIVFB (SpoIVFB-GFP; see
Materials and Methods), convert the inactive transcription factor
pro-K to the active transcription factor
K (21, 22). The additional induction of
SpoIVFA stimulates the processing reaction and is correlated with an
increase in the level of SpoIVFB-GFP (22). (The increase in
the level of SpoIVFB-GFP and the stimulation of the processing reaction
are seen only when the spoIVF operon with the
spoIVFB-gfp gene fusion substituted for wild-type
spoIVFB is expressed, not when spoIVFA and
spoIVFB-gfp are expressed independently during growth
[22a].) The additional induction of BofA blocks
processing of pro-K, in a SpoIVFA-dependent manner,
without affecting the accumulation of SpoIVFB-GFP (22).
Therefore, BofA and SpoIVFA are the only sporulation proteins needed to
inhibit SpoIVFB-GFP-mediated processing, and inhibition is exerted at
the level of the function of SpoIVFB (22).
How does BofA inhibit processing of pro-K? Using the
vegetative processing system, I show that (i) in cells engineered to produce BofA, the level of the SpoIVFA protein is increased by greater
than 20-fold; (ii) enhanced accumulation of the SpoIVFA protein does
not require SpoIVFB-GFP; (iii) stabilization of SpoIVFA by BofA
accounts for the accumulation of SpoIVFA; and, finally, (iv) a null
mutation in the ftsH gene (an ATP- and
Zn2+-dependent protease) facilitates the accumulation of
the SpoIVFA protein in the absence of BofA and substantially inhibits
pro-K processing without affecting the accumulation of
SpoIVFB-GFP. Together, these results suggest that BofA-mediated
inhibition of pro-K processing is exerted by altering
the level of the SpoIVFA protein.
| |
MATERIALS AND METHODS |
Strains, growth, and media.
The B. subtilis
strains used in this study are as follows: OR9 (PY79, wild-type
B. subtilis), OR910 [trpC2
sigK::pOR286 Pxyl-sigK (spc)
spoIIE
::kn], OR918 [trpC2
sigK::pOR286 Pxyl-sigK (spc) spoIIE::kn
amyE::Pxyl-spoIVFA, spoIVFB-gfp
(cm)], OR920 [trpC2 sigK::pOR286
Pxyl-sigK (spc),
spoIIE::kn
amyE::Pxyl-spoIVFA (cm spc)], OR948
[thrC::Pxyl-bofA (erm
phleo)], and OR956 [trpC2 sigK::pOR286 Pxyl-sigK (spc),
spoIIE::kn
thrC::Pxyl-bofA (erm
phleo), amyE::Pxyl-spoIVFA, spoIVFB-gfp (cm)] (22). The in-frame
spoIVFB-gfp gene fusion used in the above-described strains,
and those described below, was made by fusing the coding sequence for
green fluorescent protein (GFP) from Aquorea victoria to the
3' terminus of spoIVFB (21, 22). This
construction created a protein fusion at the fifth amino acid from the
COOH terminus of SpoIVFB to GFP (21, 22). The
Escherichia coli strains used in this study were DH5
(Life Technologies, Inc.), BL21(DE3)pLysS (Novagen), and OR692 (SAE97) (1). OR692 is identical to strain SAE92 [RL1196,
BL21(DE3)pSA32] (21), except that it also contains the
plasmid pLysS(cm).
For the routine growth of B. subtilis and E. coli
at 37°C, Luria-Bertani (LB) medium was used (18). To
induce promoters under xylose control during growth,
D-xylose was added to a final concentration of 20 mM when
the cultures were at an optical density at 600 nm of 0.25 to 0.30.
For sporulation assays, B. subtilis strains were grown for
23 h at 37°C in DS medium (9) supplemented with 20 mM
D-xylose and 50 µg of threonine per ml. The cells were
serially diluted, and 0.1 ml was plated on LB agar (viable cells),
while the remainder was incubated at 80°C for 20 min and then 0.1 ml
was plated on LB agar (heat-resistant cells). Colonies were counted
following an overnight incubation at 37°C.
For growth under conditions of osmotic stress, B. subtilis
strains were grown at 37°C to mid-log phase (optical density at 600 nm of about 0.5) in LB medium supplemented with 20 mM
D-xylose. The cultures were split, and NaCl was added to a
final concentration of 1.2 M to one portion and an equal volume of
H2O was added to the second portion. The growth of all the
cultures was monitored at 37°C for 9 h.
Antibiotics were used at the following concentrations for selection in
B. subtilis: chloramphenicol at 5 µg/ml; erythromycin and
lincomycin (MLS) at 1 and 25 µg/ml, respectively; spectinomycin at
100 µg/ml; kanamycin at 10 µg/ml; and tetracycline at 10 µg/ml. For selection in E. coli, chloramphenicol was used at 25 µg/ml and ampicillin was used at 100 µg/ml. To inhibit protein
synthesis in B. subtilis (see Fig. 3) chloramphenicol was
used at 200 µg/ml and was added 60 min after induction by xylose (see above).
Amylase activity was examined by growing cells overnight on 1% starch
plates (Sigma 9765) and then staining the agar with Gram's iodine
solution (Sigma HT90-2-32).
Chromosomal DNA from B. subtilis was prepared as described
previously (9).
B. subtilis strains were made competent as described
previously (9); for auxotrophic strains, the necessary amino
acid supplements were added to 50 µg/ml.
Strain construction.
Strain OR958 was made by transforming
chromosomal DNA from strain OR948 [PY79 × pOR350,
thrC::Pxyl-bofA (erm phleo)]
(22) into competent cells of strain OR920, selecting for MLS resistance.
Strain OR1017 was made by transforming strain OR918 with chromosomal
DNA from strain OR866(PY79, ftsH::tet).
[Strain OR866 was made by transforming OR9(PY79) with
chromosomal DNA from strain ED04
(ftsH::tet) (5), selecting
for tetracycline resistance.] Since the ftsH locus is
physically close to the spoIIE locus on the B. subtilis chromosome, the majority of tetracycline resistant transformants had crossed out the spoIIE mutation. Strain
OR1017 was selected because it retained resistance to kanamycin,
indicating that it retained the spoIIE mutation.
Western blot analysis.
Samples of 2 ml were collected at the
indicated intervals during growth. Whole-cells extracts were prepared
as described previously (22). The protein concentrations of
cell extracts were determined by using the Bio-Rad protein assay
reagent. Portions of 11 µg of protein from whole-cell extracts
(except in the experiment in Fig. 3, lanes f to i, where 44 µg of
protein from whole-cell extracts was used) were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%
polyacrylamide) and electroblotted to an Immobilon-P membrane
(Millipore). The blots were processed for immunodetection as described
previously (22), except that 5% dry milk was used for
blocking. Two different methods were used to detect the binding of the
primary antibody: a chemiluminescence detection system (Amersham) and a
colorimetric detection system (ProtoBlot Western blot AP system;
Promega); unless noted, the chemiluminescence system was used.
To estimate the fold difference in the accumulation of SpoIVFA in
extracts from strains OR918 and OR956, Western blot analysis was
performed with extracts from strains OR956 and OR918, 60 min after
induction with xylose. The cell extract from OR956 was diluted to
produce a signal of similar intensity to the extract from OR918 (data
not shown).
Antibodies used were rabbit polyclonal antisera against SpoIVFA
(affinity purified), K (polyclonal antiserum
[21]), or GFP (polyclonal; Clontech no. 8361-2). The
antibodies were used at 1/250, 1/10,000, and 1/2,000 dilutions, respectively.
Polyclonal antiserum against SpoIVFA was affinity purified, following
heat treatment at 50°C for 30 min, with His6-SpoIVFA from
strain OR692 as described previously (21). A detailed
protocol for the affinity purification of the serum is available upon
request. The histidine-tagged fusion protein, which contains the
COOH-terminal 116 amino acids of SpoIVFA, was purified on
Ni-nitrilotriacetic acid spin columns (Qiagen) by a denaturing method.
Image quantification.
For half-life determinations, images
were quantified with NIH Image V1.61. The program was developed at the
National Institutes of Health and is available on the World Wide Web
(19a).
Image processing.
Images were copied on an Epson 636 scanner, processed with Adobe Photoshop and Deneba Canvas on a
Macintosh PowerPC, and printed on a FUJIX3000 printer.
| |
RESULTS |
BofA-mediated inhibition of pro-K processing during
growth is correlated with an enhanced accumulation of SpoIVFA.
It
was previously shown that vegetative B. subtilis cells
engineered to produce pro-K do not process
pro-K to K (Fig.
1, top, lanes a to e). However, cells
engineered to produce pro-K, SpoIVFA, and SpoIVFB-GFP
process pro-K to K efficiently (lanes f
to j). The additional induction of the gene encoding BofA substantially
inhibits the processing reaction (lanes k to o) (22). (The
faster-migrating protein evident in lane o is an antibody-reactive
species unrelated to K.) BofA was previously inferred to
inhibit the activity of SpoIVFB-GFP because there is no obvious
difference in the level of SpoIVFB-GFP in cells that process
pro-K to K compared to cells in which
this reaction is inhibited (Fig. 1, middle, lanes f to j versus lanes k
to o) (22). Here I extend those results and show that,
surprisingly, there is a greater than 20-fold difference in the
accumulation of SpoIVFA in extracts from cells that process
pro-K to K compared to cells in which the
processing reaction is inhibited (Fig. 1, bottom, lanes f to j versus
lanes k to o) (see Materials and Methods for how the difference in the
level of SpoIVFA in cell extracts from the two strains was estimated).
(The faster-migrating protein seen in lanes a to o is an
antibody-reactive species unrelated to SpoIVFA.) These results show
that induction of the gene encoding BofA is correlated with enhanced
accumulation of SpoIVFA and inhibition of pro-K
processing.
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FIG. 1.
BofA-mediated inhibition of pro-K
processing during growth is correlated with an enhanced accumulation of
the SpoIVFA protein. Western blots of cell extracts from B. subtilis cells are shown. The proteins and protein fusion shown
above the blots were induced during growth, and cell extracts were
analyzed with antibodies that recognize pro-K and
K (top panel, colorimetric detection), GFP (middle
panel), or SpoIVFA (bottom panel). SigK refers to the
pro-K protein, and SpoIVFB-G refers to the SpoIVFB-GFP
protein fusion. Lowercase letters below the blots refer to individual
lanes. K is identified by a black star. The first lane
in each set of five corresponds to the zero time point, the second lane
corresponds to 30 min after induction, and each subsequent lane
corresponds to an additional 30 min of induction. For specific details
of the induction protocol, see Materials and Methods. Lanes correspond
to strains: a to e, OR910; f to j, OR918; k to o, OR956.
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BofA is sufficient to enhance the accumulation of SpoIVFA during
growth.
Figure 2 (lanes f to j)
shows that the enhanced accumulation of SpoIVFA evident in extracts
from cells engineered to produce pro-K, SpoIVFA, and
BofA is not seen in extracts from cells engineered to produce
pro-K and SpoIVFA alone (lanes a to e). Therefore,
during growth, overexpression of BofA is sufficient to substantially
enhance the accumulation of SpoIVFA. A further very modest enhancement
is seen in extracts from cells which additionally produce SpoIVFB-GFP
(compare lanes f to j with lanes k to o). Finally, the low accumulation
of SpoIVFA seen in Fig. 1 (bottom, lanes f to j) and 2 (lanes a to e)
is not detectably affected by the presence or absence of SpoIVFB-GFP. Therefore, SpoIVFB-GFP (a putative Zn2+-dependent
endopeptidase [14a, 16]) is not involved in
maintaining the low level of SpoIVFA.
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FIG. 2.
Induction of the gene encoding BofA is sufficient to
enhance the accumulation of SpoIVFA during growth. A Western blot of
cell extracts from B. subtilis cells is shown. The proteins
and protein fusion shown above the blot were induced during growth
(details were given in the legend to Fig. 1), and cell extracts were
analyzed with an antibody that recognizes SpoIVFA. Lanes correspond to
strains: a to e, OR920; f to j, OR958; k to o, OR956.
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In these cells, spoIVFA is not under the control of its
natural sporulation-specific promoter but is transcribed by
PxylA. A simple explanation for the enhanced
accumulation of SpoIVFA in cells engineered to produce BofA is that
BofA increases the expression of the promoter
PxylA. 
-Galactosidase assays showed that
expression of a transcriptional fusion of E. coli lacZ to
PxylA and a 5' portion of the spoIVFA
gene was similar in the presence and absence of BofA (data not shown). Therefore, the observed difference in the level of the SpoIVFA protein
(Fig. 1 and 2) is not due to transcriptional regulation of
PxylA and must be due to a postinitiation event.
SpoIVFA accumulation during growth is regulated at the level of
protein stability.
To test whether the difference in SpoIVFA
accumulation in the presence and absence of BofA is regulated by
proteolysis, pro-K, SpoIVFA and SpoIVFB-GFP were induced
in cells in which BofA was additionally induced (strain OR956) or not
induced (strain OR918), and the turnover of SpoIVFA was compared in the
two strains by blocking protein synthesis and monitoring the
disappearance of the protein by Western blot analysis. As seen in Fig.
3 (lanes f to i), SpoIVFA was degraded
rapidly in the absence of BofA with a half-life of <1.5 min. In
contrast, in the presence of BofA, the SpoIVFA protein was stabilized
with a half-life of 10 min (lanes a to e). These results show that
SpoIVFA was unstable and that proteolysis of SpoIVFA was substantially
inhibited by the presence or expression of BofA.
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FIG. 3.
BofA protects SpoIVFA from degradation. Turnover of
SpoIVFA in the presence or absence of BofA. A Western blot analysis of
the disappearance of SpoIVFA in strains OR918 and OR956 following
blocking of protein synthesis with chloramphenicol is shown. Cells were
induced, chloramphenicol was added after 60 min to inhibit protein
synthesis, and samples were taken at the times (in minutes) indicated
above the blot. Lanes correspond to strains: a to e, OR956; f to i,
OR918.
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A null mutation in a gene encoding a protease is sufficient to
enhance the accumulation of SpoIVFA and inhibit pro-K
processing during growth.
SpoIVFA is an integral membrane protein
(21). In E. coli, the essential ftsH
gene (20) encodes a membrane-bound ATP- and Zn2+-dependent protease that degrades several membrane
substrates (SecY, YccA, and subunit a of the F0
part of the H+-ATPase [12]). In B. subtilis, while the ftsH gene is essential to
sporulation, it is dispensable for growth (5, 17).
Therefore, I tested whether a null mutation of the ftsH gene
affects the accumulation of SpoIVFA during growth.
Pro-K, SpoIVFA, and SpoIVFB-GFP were induced in cells
which contain a wild-type copy (ftsH+, strain
OR918) or a null mutation (ftsH, strain OR1017) of the ftsH gene, and the accumulation of SpoIVFA was compared in
extracts from the two strains by Western blot analysis. Figure
4 shows that the level of SpoIVFA was
markedly increased in extracts from cells that contained a null
mutation of the ftsH gene (Fig. 4, bottom, lanes c and d)
compared to extracts from cells that contained the
ftsH+ gene (bottom, lanes a and b). As a
control, the accumulation of another integral membrane protein,
SpoIVFB-GFP (21), was not affected by a null mutation of the
ftsH gene (middle, compare lanes a and b to lanes c and d).
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FIG. 4.
A null mutation in the gene encoding FtsH facilitates
the accumulation of the SpoIVFA protein and inhibits
pro-K processing. Western blots of cell extracts were
analyzed with an antibody that recognizes pro-K and
K (top panel, colorimetric detection), GFP (middle
panel), or SpoIVFA (bottom panel). ftsH+ refers
to the wild-type ftsH gene, ftsH
refers to a null mutation of the ftsH gene. Lanes: a and c,
(30 min postinduction); b, d, and e, 120 min postinduction. Lanes
correspond to strains: a and b, OR918; c and d, OR1017; e, OR956.
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The level of SpoIVFA seen in the ftsH mutant strain at 120 min postinduction (Fig. 4, bottom, lane d) was comparable to that seen
in extracts from cells that contain a wild-type copy of the ftsH gene but, additionally, induced BofA (lane e). As
described above, in these cells an enhanced level of SpoIVFA is
correlated with an inhibition of pro-K processing
(22) (Fig. 1, lanes k to o). Therefore, I investigated whether a null mutation in the ftsH gene, in the absence of
BofA, would similarly affect pro-K processing during
growth. Western blot analysis with anti-K antibodies
showed that pro-K processing was substantially reduced
in extracts from cells that contained a null mutation of the
ftsH gene (Fig. 4, top, lanes c and d) compared to extracts
from cells that contained the ftsH+ gene (lanes
a and b). SpoIVFB-GFP levels were similar in the presence and absence
of FtsH (middle, lanes a to d). Consistent with the Western blot
analysis, plate tests showed that expression of lacZ fused
to a sporulation gene (gerE) under the control of K was also substantially reduced in cells that contained
a null mutation of the ftsH gene compared to cells that
contained the ftsH+ gene (data not shown).
Therefore, during growth, expression of SpoIVFA could inhibit
pro-K processing and is inferred to do so by regulating
the activity of SpoIVFB-GFP. BofA-mediated inhibition of
pro-K processing may be more efficient than a null
mutation of the ftsH gene since no K was
visible up to 4 h after induction of the gene encoding BofA (22), while in extracts from cells that contain a null
mutation of the ftsH gene, a small amount of
K was seen 120 min postinduction (Fig. 4, top, lane d).
Induction of BofA during growth does not inhibit sporulation and
does not result in a salt-sensitive growth phenotype.
The finding
that the level of SpoIVFA was markedly increased in extracts from cells
that contained a null mutation of the ftsH gene suggested
that BofA might act by inhibiting the activity of FtsH. FtsH is
required for both spore formation and growth under conditions of
osmotic stress (5, 17). Therefore I tested whether induction
of BofA during growth inhibited spore formation or affected growth
under conditions of osmotic stress. As can be seen in Table
1, induction of BofA during growth
(strain OR948) did not affect the viability of the culture or spore
formation as compared to wild-type cells (PY79). In contrast, a null
mutation of the ftsH gene (strain OR866) affected the
viability of the culture and the remaining cells did not sporulate.
To test whether the induction of BofA during growth resulted in a
salt-sensitive phenotype, exponentially growing cells were challenged
with 1.2 M NaCl and their growth was monitored in the presence or
absence of salt. As can be seen in Fig. 5
(left), the induction of BofA (strain OR948) did not affect the growth of the culture compared to that of wild-type cells (PY79). In contrast,
and consistent with previous reports (5, 17), cells containing an ftsH null mutation (strain OR866) grew at the
same rate as wild-type cells but did not reach the same density.
Wild-type cells and cells in which BofA was induced responded to
osmotic shock by resuming growth (Fig. 5, right), while cells
containing an ftsH null mutation did not resume growth
effectively. These results, and those described above, are consistent
with the conclusion that the induction of BofA during growth does not
result in an ftsH phenotype.
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FIG. 5.
Induction of BofA during growth does not result in a
salt-sensitive growth phenotype. Growth of wild-type cells (PY79) or
cells containing PxylA-bofA (OR948) or
ftsH::tet (OR866) in the presence or
absence of NaCl is shown. For details, see Materials and Methods. NaCl
was added at the zero time point depicted on these graphs. Symbols:
open squares, PY79; crossed diamonds, OR948; solid circles, OR866.
OD600, optical density at 600 nm.
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DISCUSSION |
I have investigated BofA-mediated inhibition of
pro-K processing in the absence of sporulation. The most
important finding of the present study is that BofA overexpression is
associated with a greater than 20-fold increase in the accumulation of
the SpoIVFA protein. This finding is consistent with the previous demonstration that BofA-mediated inhibition of processing requires SpoIVFA (22). Herein I have also shown that in the presence of BofA, the increased level of SpoIVFA is correlated with a
substantial inhibition of the otherwise rapid degradation of SpoIVFA.
In addition, I have shown that enhanced accumulation of the SpoIVFA
protein, achieved through the use of an ftsH null mutation
in the absence of BofA, is concurrent with inhibition of
pro-K processing. The level of SpoIVFB-GFP is not
affected, suggesting that SpoIVFA inhibits the activity of SpoIVFB-GFP.
A simple interpretation that encompasses the results presented in this
study is that BofA protects SpoIVFA from proteolysis by promoting
the assembly and maintenance of a membrane complex composed of BofA,
SpoIVFA, and SpoIVFB-GFP. Since all three proteins are integral
membrane proteins, such a complex would be membrane bound (3, 21,
23, 26). In this view, uncomplexed SpoIVFA would be subject to
proteolysis. This would be reminiscent of the degradation of
uncomplexed SecY by FtsH in E. coli (13). The
finding that a null mutation in the gene encoding FtsH enhances the
level of SpoIVFA in the absence of BofA suggests that FtsH, either
directly or indirectly, regulates SpoIVFA degradation during growth.
BofA could increase the level of SpoIVFA by inhibiting the activity of
FtsH and/or another protease or by interacting with SpoIVFA, thereby
protecting it from proteolysis. Since the induction of BofA does not
result in an ftsH phenotype, it seems unlikely that BofA
inhibits the activity of FtsH.
The pro-K processing complex in sporulating cells is
believed to be held inactive by the regulators BofA and SpoIVFA for
about 1 h (3, 4, 10, 22, 23). While it is possible that BofA protects SpoIVFA from proteolysis only during vegetative growth
(the conditions used in this study), the genetics of the phenotypes of
bofA mutants are consistent with such a role for BofA during
sporulation also. Extracts from sporulating cells that contain null
mutations in bofA have lower levels of the SpoIVFA protein
than do extracts from wild-type cells (21, 22b). Also, extracts from sporulating cells that contain specialized mutations in
spoIVFA (bofB5 and bofB8) (3,
4) have lower levels of the SpoIVFA protein than do extracts from
wild-type cells (21, 22b), suggesting that these mutant
proteins might not interact effectively with BofA and/or SpoIVFB.
Consistent with this suggestion, pro-K processing occurs
about 1 h earlier in the above-described strains than in wild-type
sporulating cells (3, 4).
Does FtsH regulate the level of SpoIVFA in sporulating cells? It has
recently been shown that FtsH does persist during sporulation (27), but there is presently no evidence that FtsH is
directly involved in the K pathway during sporulation.
FtsH is required for at least two sporulation-specific events that
occur prior to the synthesis of SpoIVFA (1a, 5, 17);
therefore, this hypothesis has not been tested directly in sporulating
cells. Principal challenges for the future will include elucidating the
molecular mechanism(s) of action of BofA and SpoIVFA.
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ACKNOWLEDGMENTS |
Strains OR958, OR866, and SAE97 were made in the laboratory of R. Losick (Harvard University). I thank P. Stragier for pDG795; W. Schumann and E. Deuerling for chromosomal DNA from strain ED04; S. Alper for strain SAE97; members of the Weisberg laboratory and the
vegetable data club at the National Institutes of Health and D. Garsin,
R. Losick, and D. Rudner for many helpful comments and suggestions; R. Weisberg and the National Institute of Child Health and Human
Development for hospitality; and A. Decatur, L. Duncan, S. Gottesman,
J. Nodwell, P. Stragier, and R. Weisberg for critical reading of the manuscript.
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FOOTNOTES |
*
Mailing address: NICHD-LMG, Building 6B-304, National
Institutes of Health, 6 Center Dr., MSC 2785, Bethesda, MD 20892-2785. Phone: (301) 496-5663. Fax: (301) 496-0243. E-mail:
resnekov{at}biosun.harvard.edu.
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Journal of Bacteriology, September 1999, p. 5384-5388, Vol. 181, No. 17
0021-9193/99/$04.00+0
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Abstract
Introduction
