Membrane topology analysis of the Bacillus subtilis BofA protein involved in promoK processing
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Microbiology (1997), 143, 1053-1 058 Printed in Great Britain
Membrane topology analysis of the Bacillus
subtilis BofA protein involved in promoK
processing
Mario Varcamonti,' Rosangela Marasco,' Maurilio De Felice3
and Margherita Sacco4
Author for correspondence: Margherita Sacco. Tel: +39 81 7257219. Fax: +39 81 5936123.
e-mail: sacco@iigbna.iigb.na.cnr.it
~
lstituto di Scienze The Bacillus subfilis BofA protein is involved in regulation of pro-# processing
dell'Alimentazione, in the mother cell during the late stages of sporulation. A computer analysis of
Consiglio Nazionale delle
Ricerche, via Roma, 83100 the BofA amino acid sequence indicates that it is an integral membrane
Avellino, Italy protein. To determine the membrane topology of the protein, a series of gene
Dipartimento di Chimica, fusions of bofA with lacZ or phoA reporter genes in Escherichia coli were
Universita degli Studi di analysed. A BofA topological model with two membrane-spanningsegments,
Salerno, via Ponte Don and with the N- and the C-terminal domains located in the region between the
Melillo, 84084 Fisciano
(Sa), Italy inner and outer membranes surrounding the forespore is presented. The
analysis of different modifications of the last five amino acid residues of the
Sezione di Microbiologia,
Dipartimento di Fisioiogia BofA protein, obtained by PCR site-directed mutagenesis, suggests a possible
Generale ed Ambientale, role of the C-terminal domain in the regulation of pro-# processing.
Universita Federico II, via
Mezzocannone 16, 80134
Naples, Italy
Keywords : sporulation, p r o 2 processing, gene fusion, mutagenesis, Bacillus subtilis
lstituto lnternazionale di
Genetica e Biofisica,
Consiglio Nazionale delle
Ricerche, via G. Marconi
10, 80125 Naples, Italy
INTRODUCTION pathways operating at the level of control of the specific
protease activity (Kaiser & Losick, 1993).
Sporulation in Bacillus subtilis involves the formation of
two cell types, the forespore and the mother cell, which, Processing of pro-aK in the mother cell is prevented by
at completion of the sporulation cycle, lyses to release mutations in the spoIVFB gene, whose product, expres-
the spore. The two compartments are generated by the sed in the mother cell under the control of aE, is the pro-
asymmetric positioning of the sporulation septum, and aK specific protease or its regulator (Cutting et a/., 1990).
later, as sporulation proceeds, the membrane of the Two other aE-controlledgenes, 6 0 f A and spoIVFA, are
mother cell migrates around the forespore, leading to involved in the regulation of the pro-aK maturation
the small compartment surrounded by two membranes (Cutting et a/., 1991b; Ireton & Grossman, 1992; Ricca
with opposite orientation. Four compartment-specific et al., 1992). Their gene products, BofA and SpoIVFA,
sigma (a) factors are known to be required during exert a temporal control on the pro-aK processing by
sporulation (Losick & Stragier, 1992; Errington, 1993): negatively regulating the SpoIVFB activity (Cutting et
their expression is regulated at different levels, the al., 1990;Ricca et al., 1992). Processing of pro-aK is also
ultimate and most determinant control of their ex- controlled by a forespore-specific gene product, SpoIVB,
pression being exerted at the level of their activity. In the expressed under the control of the forespore aG factor
mother cell compartment two a factors are successively (Van Hoy & Hoch, 1990; Cutting et al., 1991a). The
active, aE and aK. Both are expressed as pro-a factors, SpoIVB protein releases the inhibitory effect of BofA and
bearing N-terminal 29 (aE) and 20 (aK) amino acid SpoIVFA on the SpoIVFB protease (Cutting et a/.,
prosequences (Jonas et al., 1988; Stragier et al., 1988, 1991b; Gomez et al., 1995). Mutations in either 60fA or
1989; Kroos et al., 1989). Processing of these two pro-a spolVFA lead to bypass of the forespore (Bof) signal
factors depends on intercellular signal-transduction (Cutting et al., 1991b; Ricca et al., 1992). SpoIVFA,
SpoIVFB and BofA are inferred to be membrane-
................................................................................................................................................. spanning proteins, located in the mother cell membrane
Abbreviations: AP, alkaline phosphatase; P-Gal, P-galactosidase. surrounding the forespore (Cutting et al., 1991b; Ricca
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et al., 1992; Kaiser & Losick, 1993), while SpoIVB Table 1. Two series of primers were designed, which were
would be secreted from the forespore into the space characterized by the different frame of the BamHI site needed
between the two membranes (Cutting et al., 1991a; for the cloning upstream of the phoA and lac2 reporter genes.
Amplifications were carried out using each primer paired
Kaiser & Losick, 1993).
with the Pspac primer (5'-CCTCTAGAGGCGTATCAC-
Knowledge of the membrane topology of SpoIVFA, GAGGCCC-3'), containing the X6aI restriction site. For the
in-frame fusions with the phoA or lacZ genes, the appropriate
SpoIVFB and BofA will help in the understanding of
PCR products were digested with X6aI/BamHI and
regulation of pro-oK processing. We report here a EcoRI/BamHI and cloned in plasmids pMVlOO and pNM482
membrane topology analysis of the BofA protein by (Minton, 1984), respectively. A restriction site for EcoRI,
means of 6ofA-lac2 and 6ofA-phoA fusions. Based on utilized for the 6ofA-lac2 fusions, was present on plasmid
site-directed mutagenesis and deletion analysis we hypo- pER76 about 50 bp upstream of the Pspac promoter. The
thesize that the last three C-terminal amino acid residues resulting plasmids carrying the 6ofA-phoA and 6ofA-lac2
of the BofA protein, belonging to a domain located in fusions (Table 1) were used to transform the E. coli strains
the region between the two membranes surrounding the CC118 and TG1, respectively. The fusion junction of each
forespore, are involved in the inhibitory effect on plasmid was verified by DNA sequencing, utilizing oligo-
SpoIVFB function. nucleotides 5'-CGGGAAAGGTTCCGTCC-3' and 5'-
GGGTTTTCCCAGTCACG -3' for the 6ofA-phoA and the
6ofA-lacZ fusions, respectively.
METHODS Cell fractionation. E. coli cells carrying the 6ofA-phoA fusions
were fractionated into periplasmic, cytosolic, and membrane
Bacterial strains. The E. coli strains CC118 [araD139 A(ara, components (modified from Neu & Heppel, 1965). Late-
leu)7697 AfacX74 phoAA20 galE galK thi rpsE argE(Am) exponential-phase cells from 30 ml cultures were recovered by
recAI] (Manoil & Beckwith, 1985) and TG1 (supE hsdA5 thi centrifugation and osmotically shocked. The spheroplasts
Ah-proBA) (Amersham) were used for analysis of alkaline were sedimented by centrifugation, and the supernatant,
phosphatase (AP) and b-galactosidase @-Gal) activities, re- representing the periplasmic fraction, was concentrated by
spectively. B. subtilis strain MVlOlO (spoIIIG A60fA) was centrifugation in a Centricon 10 unit (10000 Da molecular
obtained by marker replacement of the 6ofA gene with an erm mass cutoff, Amicon). The spheroplasts were resuspended in
cassette in strain SC500 (spoIIIG) (Cutting et al., 1990). 10 mM Tris/HCl p H 8.0 and sonicated on ice. The samples
MVlOlO was utilized for the complementation analysis of were then spun at 3000 g for 10 min at 4 "C to remove cellular
6ofA mutations. The standard rich medium was T Y (Sam- debris, and the supernatant was centrifuged at 140000g in a
brook et al., 1989). When required, ampicillin was added to a Beckman SW 50.1 rotor for 90 min. The resulting supernatant
final concentration of 50 pg m1-l. was the cytoplasmic fraction and the pellet contained the
membrane fraction.
General methods. B. subtilis competent cells were prepared
and transformed by the methods of Dubnau & Davidoff- Detection of AP activity in polyacrylamide gels. Proteins in
Abelson (1971). When required, chloramphenicol or erythro- the three cellular fractions were separated by SDS-PAGE
mycin were added to agar plates to concentrations of 5 or (12%), as described by Laemmli (1970). The gel was fixed
1 pg ml-', respectively. Sporulation was induced on Difco immediately after the run by a 3 h treatment in 20% (v/v) 2-
sporulation agar at 37 "C. The specific activity of P-Gal was propanol in 1 0 m M Tris/HCl (pH7.5) and washed with
determined as described by Miller (1972) with the substrate 10 mM Tris/HCl (pH 7.5) for 12 h. The gel was incubated at
ONPG. 37 "C in 10 ml AP buffer (100 mM Tris/HCl, 100 mM NaCl,
50 mM MgCl,, pH 9.3, 66 pl NBT (nitro blue tetrazolium)
DNA methodology. Standard molecular cloning techniques and 33 pl BCIP (5-bromo-l-chloro-3-indolylphosphate), until
were performed as described by Sambrook et al. (1989). PCRs the blue colour which indicates PhoA activity had developed.
were carried out with Taq DNA polymerase (Promega)
according to the manufacturer's recommendations. Nucleo- AP assay. Bacteria in 800 pl late-exponential phase culture
tide sequences were determined by the procedure of Sanger et were permeabilized with 20 p1 chloroform and 20 pl 0.1 O/O
al. (1977), with a T 7 DNA polymerase sequencing kit SDS. Para-nitrophenylphosphate ( 0 1 ml of a 10 mg ml-l
(Pharmacia) and specific oligonucleotides as sequencing pri- solution in 1 M Tris p H 9.0) was added, and the mixture was
mers. incubated at 37 "C until yellow colour had developed.
Reaction was stopped with 0.1 ml N a O H (10 M). Specific
Plasmid constructions. The pMVlOO plasmid was constructed activity was calculated according to the formula :
by cloning the 2.6 kb HindIII-XhoI fragment from the p P H 0 7 1000 x A,,,/[reaction time (min) x volume (ml) x OD,,, of
plasmid (Gutierrez & Devedjian, 1989), carrying the phoA culture] (Brockman & Heppel, 1968).
gene, into the HindIII-XhoI digested pJRD184 (Heuster-
spreute et al., 1985). A 330 bp HindIIIISphI fragment, PCR site-directed mutagenesis. Plasmid pER44 (Ricca et al.,
spanning from the - 10 to the terminator region of the 6ofA 1992), containing the entire 6ofA gene and 280 bp upstream of
gene, was cloned downstream of the Pspac promoter of the 60fA promoter, was digested with ScaI to generate a linear
pAG58 (Jaacks et al., 1989) to yield pER76. template for the PCR mutagenesis reaction. The primer
corresponding to the 5' of the 60fA gene consisted of the E4
Constructionof gene fusions using PCR. Using plasmid pER76 oligonucleotide (Ricca et al., 1992) containing an EcoRI site.
as template, DNA fragments, spanning 100 bp upstream of the The primers corresponding to the 3' end of the gene,
Pspac promoter and various portions of the 60fA coding containing a BamHI site, were designed to introduce muta-
region, were generated by PCR, using the primers shown in tions leading to individual Lys83Gln, Lys83amber, Gln84Leu,
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PheSSSer, Phe85ochre, Ile86Thr, Ile87Ser and
IleIle86/87MetPhe amino acid substitutions in BofA. The
resulting 600 bp PCR fragments were isolated from the gel and
cloned into pDG364 (used as an amyE insertion vector) m3
+ @
V P I ~ ~ L V T T A I S GGI I P G I A A L V VQFll
~
(Cutting & Vander Horn, 1990), after an EcoRIIBamHI
double digestion. The presence of the expected mutations in
the amplified bofA genes was verified by sequence analysis Fig. 7. Amino acid sequence of BofA (Ricca e t a/., 1992).
(data not shown). The resulting plasmids were linearized, gel Individual fusions are indicated by a. The putative membrane-
purified, and used to transform the B. subtilis MVlOlO spanning segments (TMI-3) are boxed. Charged amino acid
(spoZZZG AbofA) strain. The chloramphenicol-resistant trans- residues are indicated by and -. +
formants were tested for their a m y phenotype to verify that
the insertion had occurred by marker replacement (double)
recombination at the amyE locus.
RESULTS Specific PCR fragments amplified from DNA template
(pER76), and containing different portions of the 6ofA
Construction of bofA-phoA and bofA-lacZ fusions gene, were digested with X6aIIBamHI and cloned into
It has been previously proposed that the three hydro- the multiple cloning site of pMVlOO to generate
phobic stretches of amino acid residues identified in the 6ofA-phoA fusions encoding amino acid residues 1-29
BofA protein may correspond to membrane-spanning (BofA,,,,), 1-58 (BofA,,,,), 1-78 ( h f A ~ , , p )and 1-87
domains (Ricca et al., 1992). We identified the positions (BofATI1,P) of BofA upstream of the PhoA protein (Table
of the putative membrane-spanning segments of the 1, Fig.'i)-(see Methods). The junction of each fusion was
BofA protein by using the TopPred I1 computer program confirmed by nucleotide sequencing (data not shown).
(Claros & von Heijne, 1994). The computer model The point of fusion of the hybrid proteins occurred at
predicted three membrane-spanning domains, with the the two putative hydrophilic loops (BofA,,,,,
N-terminus domain located in the space between the BofA,,,,), within the TM3 putative intramembrane
two membranes surrounding the forespore, and the C- region (BofA,,,,), and at the C-terminus of the BofA
terminus domain located in the mother cell cytoplasm. protein (BofA,,,,) (Fig. 1). Parallel experiments were
performed to construct 6ofA-lac2 fusions, to yield
The predicted three-transmembrane topological model BofA-p-Gal hybrid proteins named BofAW2,,,
of the BofA protein was investigated by constructing a BofA,,,,, BofA,,,, and BofA,,,, (Table 1).
series of 6ofA-phoA and 6ofA-lac2 gene fusions. This
approach is based on the observation that AP is active The E. coli strains CC118 (phoAA20) and T G 1
when exported across the cell membrane, but exhibits (Alac-proBA) were transformed with plasmids carrying
low activity in the cytoplasm (Derman et al., 1993). O n the 6ofA-phoA and 6ofA-lac2 fusions, respectively. AP
the contrary, p-Gal hybrids express activity only if the p- and p-Gal enzyme activities were assayed during expo-
Gal moiety is retained in the cytoplasm (Slauch & nential growth. AP activity was found in strains bearing
Silhavy, 1991). Hence, in-frame fusions of a gene hybrid proteins B O ~ A , ~ ,BofA,,,,
~, and BofA,,,, (Table
encoding a target membrane protein with the phoA and 2), indicating the periplasmic localization of their PhoA
the l a c 2 genes can differentiate between regions enco- moiety. In a reciprocal way, p-Gal activity was found
ding cytosolic or periplasmic domains on the basis of the only in the strain producing BofA,,,,, indicating the
different levels of AP and /?-Gal activities (Manoil & cytoplasmic localization of the p-Gal moiety in the
Beckwith, 1986; Slauch & Silhavy, 1991). hybrid protein (Table 2).
Table 7. BofA fusion primers
I Sequence (5' -,3'):' Cloning vector Resulting plasmidt Hybrid protein+
ccggcggatcCCACTTTAAAGGCTT pMVlOO pW29P
CggggggatcCACATGAATGCCAAG pMVlOO pV58P
gagttggatcCGCAGCTATTCCGGG pMVlOO pA78P
gggggggatcctcAATG AT AAATTGCTT pMVlOO pI87P
ggggatccCCACTTTAAAGGCTT pNM482 pW29L
ggggatccCACATGAATGCCAAG pNM482 pV58L
ggggatccCGCAGCTATTCCGGG pNM482 pA78L
ggggatccAATGAT AAATTGCTT pNM482 pI87L
* Upper-case letters represent 60fA residues. Bold lettering shows the BamHI linker introduced in all
amplified fragments to facilitate cloning.
t P, bofA-phoA fusions ; L, 6ofA-lac2 fusions.
*Subscripts represent the last BofA residue present in the hybrid protein.
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restores aK-directed gene expression, producing Pig'
colonies (Cutting et al., 1990). In the double mutant
Strain AP" Strain P-Galt MV1010, the introduction of a functional BofA protein
complements the A 6 o f A mutation, restoring a Pig-
CC118 3 TG1 2 phenotype (Ricca et al., 1992). In our complementation
CC118 (pW29P) 11 TGl(pW29L) 180 analysis of the A6ofA mutation in strain MV1010, the
CC118(pV58P) 157 TG1 (pV58L) 12 introduction of 6 0 f A alleles coding for proteins with
CC1lS(pA78P) 148 TGl(pA78L) 22 single or double amino acid substitutions restored a Pig-
CC118 (p187P) 134 TGl(pI87L) 12 phenotype (Table 3), indicating the presence of func-
tional modified BofA proteins. The absence of the last
five or three amino acid residues caused a loss of
function of the BofA protein, which did not complement
the A6ofA mutation, leading to retention of the Bof
(Pig') phenotype (Table 3).
DISCUSSION
Identification and cellular localization of the In this study we determined the membrane topology of
BofA-PhoA hybrid proteins the B. subtilis BofA protein, involved in the regulation of
the pro-aK processing in the mother cell during sporu-
To determine the cellular localization of the four
lation. A total of four plasmid-borne 6ofA-phoA fusions
BofA-PhoA hybrid proteins, we performed a cell frac-
and four 6ofA-lac2 fusions, which contained various
tionation experiment, followed by gel electrophoresis of
portions of the N-terminus of BofA fused in-frame to
the proteins and BCIP colorimetric assay (see Methods).
PhoA and P-Gal, respectively, were analysed. The
The cytoplasmic fractions of the four strains did not
BofA,,,, hybrid protein did not show AP activity,
show any AP activity (data not shown). In the membrane
while the BofAV5,,, BofA,,,, and BofA,,,, hybrid
fractions of strains expressing BofA,,,, and BofA,,,,,
proteins showed significantly high AP activity (Table 2).
the size of the hybrid proteins correlated well with those
inferred from the location of the fusion junctions (Fig. 2, This result was confirmed by a cell fractionation
lanes 4 and 8). In the same fractions a band cor- analysis, which showed AP activity in periplasmic and
responding to mature PhoA was also found. The membrane fractions of strains bearing BofA,,,, and
observation that an AP-sized proteolytic product may be BofA,,,, hybrid proteins, and in the periplasm of the
present in membrane and periplasmic fractions of cells strain expressing BofAAYap(Fig. 2). In a reciprocal
carrying PhoA-fusion proteins has been widely reported manner, high P-Gal activity was found in a strain
(Allard & Bertrand, 1992; Dassa & Muir, 1993; expressing the BofA,,,, hybrid protein while no P-Gal
Danielsen et al., 1995). The periplasmic fractions of activity was detectable in strains producing the
strains expressing BofAV5,,, BofA,,,, and BofA,,,, BofAV5,,, BofA,,,, and BofA,,,, hybrid proteins (Table
showed a band corresponding to the mature PhoA and 2). The two types of hybrid proteins showed comple-
an extra band with a higher molecular mass, probably a mentary properties, which have been extensively used
product of an alternative proteolytic event (Fig. 2, lanes to support membrane topological models (Manoil,
5,7 and 9). The strain expressing BofA,,,, only had AP 1990; Dassa & Muir, 1993; Hennessey & Broome-
activity in the periplasmic fraction, probably indicating Smith, 1993;Danielsen et al., 1995;Paulsen et al., 1995).
the high efficiency of proteolytic events determined by The experimental data are consistent with a topological
the particular fusion joint occurring in this hybrid model of BofA having two transmembrane segments
protein. (TM1 and TM2) and a third domain, spanning amino
acid residues 58-87, with hydrophobic characteristics of
The strain expressing BofA,,,, did not have AP activity a transmembrane domain (aa 62-82>, but with a peri-
in any fraction (Fig. 2, lanes 2 and 3), indicating that the plasmic localization (Fig. 3). An alternative hypothesis
AP moiety of the hybrid protein is not exported into the could be that the third hydrophobic domain loops into
periplasm and might be degraded in the cytoplasm, the membrane, exposing its N- and C-termini to the
probably as a consequence of its incapacity to fold periplasm. The existence of a proline residue in position
properly (Derman et al., 1993). 74 could support the latter hypothesis. In both cases the
C-terminal domain would be exposed in the region
bofA mutagenesis between the two membranes surrounding the forespore.
We performed a series of 6 o f A site-directed mutations We performed an extensive site-directed mutagenesis
of the 3' region of the gene (see Methods). The effect of analysis of the 3' end of the 6 0 f A gene, encoding five C-
each mutation was tested by insertion of the mutagenesis terminal amino acid residues. Each single amino acid
products at the amyE locus of the B. subtilis strain substitution, together with a double substitution of the
MVlOlO (spoZZZG A6ofA). The spoIIZG mutation pre- amino acid residues in positions 86 and 87, yielded a
vents aK-directed gene expression, including the ex- modified protein retaining wild-type function, as ana-
pression of the c o t A gene, which makes sporulating cells lysed by a complementation test on B. subtilis strain
brown (Pig'). In a spoZZlG background, a 6 o f A mutation MVlOlO (Table 3). Mutations leading to an amber
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Figrn2. Gel electrophoresis of cell fractions.
Detection of AP activity in membrane and
periplasmic fractions of CCl18(pW29P)
(lanes 2 and 3, respectively); CC118(pV58P)
(lanes 4 and 5); CC118(pA78P) (lanes 6 and
7) and CC118(p187P) (lanes 8 and 9). Lane 1 :
PhoA-positive control (70 units purified
enzyme). Molecular masses of PhoA, and of
hybrid proteins BofA, and BofA,,,, are
indicated.
Table 3. Complementation analysis of bofA deletion Based on the membrane topological model of BofA
presented here (Fig. 3 ) , and on a site-directed muta-
Insertion at amyE" Bof phenotypet genesis analysis we can hypothesize that the C-terminal
tail of the protein represents a functional domain that is
60fA (K83Q) either involved in correctly positioning the protein with
60fA (K83amber) respect to the heterotrimeric complex, or is important in
60fA (Q84L) the negative regulation that BofA exerts directly or
6ofA (F85S) indirectly on the SpoIVFB proteolytic activity.
6ofA (F85ochre)
6ofA (186T)
ACKNOWLEDGEMENTS
60fA (187s)
60fA (II86/87MF) W e thank F. Arigoni and E. Ricca for providing strains and
plasmids, E. Patriarca for critical reading of the manuscript,
:'Recipent strain utilized MVlOlO (spolllG ~ 6 0 f A )Amino
. acid and C. Sole and P. Villano for technical assistance. This work
substitutions and the relative positions are indicated in par- was supported by CNR, PF 'Biotecnologie e Biostrumen-
entheses. tazione ' and ' Ingegneria Genetica ' and by MIRAAF, ' Piano
t The Bof phenotype was determined by phase-contrast micro- Nazionale Biotecnologie Vegetali '.
scopy of sporulating colonies grown on DS agar for 3 d at
37 OC. Bof+ cells produced phase-grey prespores and released
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