FEMS MicrobiologyLetters 51 (1988) 109-112
Published by Elsevier
109
FEM 03194
The Bacillus subtilis spolIA locus is expressed at two times
during sporulation
H e r m i n i a de L e n c a s t r e
a
a n d P a t r i c k J. Piggot b,,
a Grupode Gendtica Molecular, lnstituto Gulbenkian de Cidncia, Oeiras, Portugal,
and b National Institutefor Medical Research, London, U.K.
Received 8 February 1988
Accepted 17 February 1988
Key words: Bacillus subtilis; spolIA locus; Sporulation
1. SUMMARY
The Bacillus subtilis spolIAA and spolIAB genes
were fused to the Escherichia coli lacZ gene on a
novel integrational plasmid vector. The constructs
were integrated into the B. subtilis chromosome,
and used to show that the spolIA locus was expressed at two times during sporulation.
scriptional spolIA-lacZ fusions to show that transcription of spolIA commences about 90 min after
the start of sporulation [5,6]. This result agrees
with the time of transcription determined by
Northern hybridization [7,8]. We have now used
translational spollA-lacZ fusions to analyze
spolIA expression, and report here the somewhat
surprising observation that expression occurs at
two times during sporulation.
2. I N T R O D U C T I O N
3. M A T E R I A L S A N D M E T H O D S
Spore formation of Bacillus subtilis is a primitive cellular differentiation. It requires, among
other things, the expression of some 50 spo loci
[1]. We have been studying the spolIA locus in
order to investigate how expression of such a large
group of genes is coordinated. The locus consists
of a polycistronic operon [2]. The putative protein
product of the third gene of the locus has extensive sequence homology with RNA polymerase
sigma factors [3,4]. We have previously used tran-
Correspondence to: P.J. Piggot, Department of Microbiology
and Immunology, Temple University School of Medicine,
Philadelphia, PA 19140, U.S.A.
3. I. Bacterial strains and plasmids
The B. subtilis strain used was MB24 metC3
trpC2 rif-2. The Escherichia coli strain was DH1
[91.
All plasmids replicated autonomously in E. coli
and were maintained in strain DH1. Plasmid
pPP81 contains a transcriptional spolIA-lacZ fusion and has been described previously [5,6].
3.2 Assays
/3-Galactosidase was assayed by the method of
Miller [10]. Results are expressed as specific activity in nmol O-nitrophenyl galactoside (ONPG)
hydrolyzed/min per mg bacterial dry weight.
0378-1097/88/$03.50 © 1988 Federation of European MicrobiologicalSocieties
110
Procedures for sporulation and transformation
have been described previously [2]. Plasmid copy
numbers were determined as described previously
[6].
4. RESULTS AND DISCUSSION
4.1. Construction of translational spoIIA-lacZ fusions
The integrative translational fusion vector used,
pPP103 (Fig. 1), was constructed by inserting the
PstI fragment from pMC1871 that carried the
lacZ gene [11] into the unique PstI site of pJH101
[12]. pPP103 determines resistance to tetracycline
and chloramphenicol in E. co#. It contains a
E
J t
~%/
.t
t
j
Pv
,I
I
1 kbp
INSERT
Ah Pv
Ah
II
IAI
!
pPP 1 1 1 J
,g
B
I
C
I
spollA
I ,
Fig. 1. Restriction endonuclease map of plasmid pPP103 and
of the spolIA region of the chromosome showing the portions
inserted into pPP103 to construct plasmids pPP111 and pPP115.
The lacZ structural gene is indicated on pPP103 by a heavy
line. The three open reading frames of spolIA are designated
A, B, and C. Direction of transciption of spolIA and of lacZ is
indicated by arrows. Restriction sites: Ah, AhaIII; E, EcoR1;
P, Pstl; Pv, PvuII; S, Smal (Ah and Pv sites are only shown
for the spollA region).
unique SmaI site suitable for the construction of
translational fusions.
Plasmid pPPl11 (Fig. 1) was constructed by
ligating a 1.8 kbp PouII fragment from priM2 [13]
to the SmaI site of pPP103; in p P P l l l , the spollAB gene is fused in phase to lacZ. Plasmid
pPPll5 (Fig. 1) was constructed by ligating a 1.05
kbp AhaIII fragment from a derivative of priM2
[5,6] to the SmaI site of pPP103; in pPPI]5, the
spolIAA gene is fused in phase to lacZ. Analysis
by digestion with restriction endonucleases confirmed that the insertions were in the correct
orientation. The 1.05 kbp AhaIII fragment used to
construct pPP115 was the same fragment as that
used to construct the transcriptional fusion in
pPP81 [5,6]. The cloned region contained all the 5'
sequence required for spolIA expression [2].
4.2. Expression of fl-galactosidase from spoIIA-lacZ
fusions during sporulation
Plasmids pPPll5, and pPP81 were introduced
into the Spo + B. subtilis strain MB24 by transforrnation for the vector-determined resistance to
chloramphenicol. Southern hybridization was used
to confirm that the plasmids had integrated into
the homologous region of the chromosome by a
Campbell-like mechanism. Purified MB24
(pPPll5) and MB24 (pPP81) clones containing a
single copy of the integrated plasmid were induced
to sporulate in modified Schaeffer's liquid sporulation medium [14] containing 3 /tg chloramphenicol per ml, and samples were taken to
assay for fl-galactosidase (Fig. 2). The strains gave
more than 80% sporulation as determined by phase
contrast microscopy. The strain with the translational fusion produced more enzyme than the
strain with the transcriptional fusion; this was
probably caused by the different strengths of the
ribosome binding sites. The two strains began to
produce enzyme at the same time, suggesting that
control of the timing of spolIA expression was
transcriptional.
With strain MB24 (pPPll5) there was a sharp
fall in fl-galactosidase activity after the initial
period of synthesis, indicating that the fused protein was unstable. The fall was then followed by a
second peak of activity. A strain with four copies
of pPP115 integrated in tandem in the chro-
111
tional fusions (Fig. 2). MB24 (pPP81) does not
show the sharp decline in activity between two
peaks that is seen with the other strains; this is
probably a consequence of greater stability of the
native fl-galactosidase as compared to fl-galactosidase fused to the spolIA proteins. Thus, the
results are consistent with two periods of transcription, one starting at about tl. s and the other
at about /4; in the system phase grey prespores
begin to appear at about t 5.
Savva and Mandelstam [7,8] have analyzed
spolIA transcription by Northern blots. A spolIA
transcript was first detected at t I and was estimated to be 1.4-1.7 kb; it reached m a x i m u m
concentration at t 2. This corresponds well to the
first period of fl-galactosidase synthesis (Fig. 2). A
second, larger transcript was present in blots of
R N A prepared at /3, t4 and t 5. It is not clear,
however, if this corresponds to the second period
of spollA expression detected with translational
fusions (Fig. 2), as the 1.4-1.7 kb transcript was
present in all samples that contain the larger tran-
00-
-300
e=
.'2_
-200
¢u
o=
.)
u
u
O9
0
2
Time (h) a f t e r s t a r t
4
6
of sporulation
Fig. 2. flmGalactosidase synthesis during sporulation for B.
subtilis MB24 having single copies of plasmids integrated into
the chromosome. Symbols: O, pPPll5 (translational fusion to
spollAA); ©, pPP8] (transcriptional fusion); 13, plasmid with
same insert as pPPll5, but in opposite orientation with respect
to lacZ. Specific fl-galactosidase activity is in nmol ONPG
hydrolyzed/min per mg bacterial dry weight; note that the
scale is changed for the second peak of enzyme activity with
MB24 (pPPll5).
-1000
._~
"~
mosome produced substantially more enzyme than
the strain with one copy (Fig. 3), but showed the
same two peaks of activity. These peaks were also
seen with an MB24 ( p P P l l l ) clone with a copy
number of 3 (Fig. 3). They were also seen in a
series of clones of MB24 ( p P P l l 5 ) and MB24
( p P P l l l ) for which the copy numbers were not
determined (data not shown). The second peak
indicates a second period of transcription, as the
spolIA m R N A is unstable with a half-life of 2 - 3
min [8]. There is an increase in fl-galactosidase
activity for the strain with the transcriptional fusion (MB24 [pPP81]) that coincides with the second burst of activity for the strains with transla-
500
g
•
._~
a
v
0
/
2
Time
!
!
4
6
I h l a f t e r t h e s t a r t of s p o r u l a t i o n
Fig. 3. fl-Galactosidase synthesis during sporulation for B.
subfilis MB24 containing multiple copies of plasmids with
translational fusion to spoIIAA (pPPll5) and spoIIAB
(pPPlll). Symbols: O, pPPll5, four copies: ©, pPPlll, three
copies. Specific fl-galactosidase activity is in nmol ONPG
hydrolyzed/min per mg bacterial dry weight.
112
script; the transcripts were unstable, at least in the
presence of actinomycin D, so that both represent
de novo synthesis [7,8].
Errington and Mandelstam [15] have recently
reported studies of a translational spolIA-lacZ
fusion using a phage-based system. They found
the same initial time of expression of spolIA, but
did not observe the second sharp peak of expression. It is striking that the maximum enzyme
activities reported here are about 20-fold higher
than those reported by Errington and Mandelstam
[15]. Consequently, we consider that the fusions
used here are a more reliable indicator of spolIA
expression. In support of this conclusion, the same
two peaks of activity were observed with the spoIIAB-lacZ fusion as with spolIAA-lacZ fusion (Fig.
3). One period of spolIA expression is definitely
necessary for spore formation [2]. The role of a
second period of expression is unclear. As both
peaks of activity are seen in a spoOJ mutant
(unpublished data), they do not depend on any of
the recognizable morphometric changes during
sporulation.
ACKNOWLEDGEMENTS
This work was supported in part by Public
Health Service grant AI23045 from the National
Institute of Allergy and Infectious Diseases, and
in part by a grant from the Gulbenkian Foundation.
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