Gene, 152 (1995) 69-74

1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50 69

GENE 08444

Expression of the subtilisin Carlsberg-encoding gene in Bacillus

licheniformis and Bacillus subtilis
(Promoter; mRNA; regulation; DegU; DegQ; target)

Myra F. Jacobs
Department of Microbiology, Technical University of Denmark, 2880 Lundtofte, Denmark. Tel. (45-45) 933-422

Received by R.E. Yasbin: 29 April 1994; Revised/Accepted: 11 July/19 July 1994; Received at publishers: 22 September 1994

SUMMARY

The cloning and sequence of the 5' untranslated region (5'-UTR) of the Bacillus licheniformis (BI) 6816 subtilisin
Carlsberg gene (subC) are reported here. The 5' and 3' ends of subC transcripts were characterized, and the promoter
identified. Expression was studied using a fused lacZ reporter gene integrated into the chromosome of heterologous
host Bacillus subtilis (Bs). 13Gal activities of mutants deleted within the promoter region identified a region which is
required for stimulation by the transcriptional activator proteins, DegU and DegQ. This region is close to the transcrip-
tion start point (tsp), and is adjacent to a sequence homologous to that involved in DegU/Q stimulation of the Bs
subtilisin gene, aprE. Expression of subC in Bs was optimized by the the use of a heterologous promoter and by the
deletion of UTR sequences predicted to be involved in secondary structures in the native subC mRNA. Sequence
comparison with other subtilisin Carlsberg-type-encoding genes revealed a high degree of conservation of the entire
5'-UTR, including regulatory sequences and promoter, as well as part of the structural gene.

INTRODUCTION
Regulation of the aprE gene, encoding the 275-aa
BPN'-type alkaline serine exoprotease (subtilisin) of Bs,
is quite complex (Strauch and Hoch, 1993). Many factors
having pleiotropic regulatory effects influence aprE
expression, acting via 5' regions in cis. In some cases,
direct contact with aprE DNA has been demonstrated,
and target sites defined, e.g., the transition-state regulator
Hpr, affecting subtilisin production negatively, interacts
with sites quite distant from the aprE transcription start
point (tsp)(Strauch and Hoch, 1993). Although their
direct contact with aprE has not been demonstrated, the
transcriptional activators DegQ and DegU stimulate
subtilisin production via target sites closer to the pro-
moter (Henner et al., 1988).
Little is known about regulation of the homologous
B1 gene, which encodes the industrially important 274-aa
Carlsberg-type subtilisin (SubC). This is primarily due to
a dearth of suitable subC clones. Also, while Bl homo-

Correspondence to: Dr. M. F. Jacobs at her present address: Department
of Microbiology, Building 231, University of Maryland, College Park,
MD 20742, USA Tel. (1-301) 405-5430; Fax (1-301) 314-9489.
Abbreviations: aa, amino acid(s); A45o, absorbance at 450 nm (1 cm);
Ap, ampicillin; aphA3, kanamycin-encoding gene; aprE, alkaline serine
protease (subtilisin)-encoding gene of B. subtilis; B., Bacillus; [3Gal,
13-galactosidase; BGSC, Bacillus Genetic Stock Center; Bl, Bacillus
licheniformis; bp, base pair(s); Bs, Bacillus subtilis; cDNA, complemen-
tary DNA; Cm, chloramphenicol; AG, Gibbs free energy; kb, kilobase(s)
or 1000 bp; Lac + , phenotypically expressing lacZ; lacZ, gene encoding
!3Gal; LB, Luria-Bertani medium (1% Bacto-tryptone/0.5% yeast
extract/0.5% NaC1 pH 7.5); MSM, modified Schaeffer's medium; nt,
nucleotide(s); O, operator; p, plasmid; P, promoter; PA, polyacrylamide;
PolIk, Klenow (large) fragment of E. coli DNA poIymerase I; R, resis-
tant/resistance; RBS, ribosome-binding site(s); sacB, levansucrase-
encoding gene; SDS, sodium dodecyl sulfate; subC, subtilisin Carlsberg
gene; t, transcription terminator(s); To, time end of exponential growth;
T1,2, etc., hours after To; tsp, transcription start point(s); UTR,
untranslated region(s); xynB, xylosidase-encoding gene; wt, wild type;
[ ], denotes plasmid-carrier state; ::, novel junction (fusion or insertion);
' (prime), denotes a truncated gene at the indicated side.

SSDI 0 3 7 8 - 1 1 1 9 ( 9 4 ) 0 0 6 5 5 - 5
70

logues of some genes known to be involved in aprE regu- a

lation have been identified, e.g., spoOH (Dubnau et al., pMJ51
pSUB3, pMJ29
1988) and degQ (Amory et al., 1987), characterized Bl pMJ57
Hybridization probe
regulatory mutants are still lacking. Bell Sacll S Stul Oral Hindlll EcoRI Bcll Haolll
Here the cloning of the Bl NCIB 6816 subC promoter Aval

and adjacent regions, and their effects on subC expression

in Bs, are described. The results validate the use of a
closely related heterologous host, in cases such as this,
where relevant regulatory alleles are not available in the
1 334 nt
organism of origin.
b qlaqI
Sau3AI TaqI Sau3AI 60
GATCGACAAG ACCGCAACCT CCTTCGACAA AAAATGATCT CTTAAAATAA ATGAATAGTA
EXPERIMENTAL AND DISCUSSION
TTTTCATAAA ATGAATCAGA TGGAGCAATC TCCTGTCATT CGCGGCCCTC GGGACCTCTT
770 iso
(a) Cloning the subC 5' flanking region TCCCTGCCAG GCTGAAGCGG TCTATTCATA C T T T C G ~ T GAACATT+T CTAAAACAGT

A clone of the Bl 6816 subC structural gene (pSUB3,
Fig. la) has previously been described (Jacobs et al.,
1985), but it did not display endogenous promoter activ-
ity. To clone the promoter and regulatory sequences
required in cis, restriction sites 5' from subC in 6816 chro-
mosomal DNA were first mapped by Southern blotting
analysis. Relevant sites are shown in Fig. la.
Attempts to clone 5'-UTR on a 2.4-kb BclI Bl DNA
fragment (Fig. la) into either E. coli or Bs hosts using a
variety of cloning strategies failed; subC-specific clones
all had deletions or rearrangements at the 5' end. One
such plasmid clone, pMJ2, obtained in low copy in E.
coli, was characterized further.
Southern blotting comparison of pMJ2 with 6816
chromosomal DNA revealed that the 5' limit of contigu-
ous Bl subC sequences lay between the closely spaced
TaqI sites, and the SacI! site (Fig. la,b). Expression
studies showed that the native subC promoter (P~,bC), as
well as regions required for regulated subC expression
(see sections b and g) were present in pMJ2, despite the
rearrangement. The subC gene was reconstituted from
material in pMJ2 and pSUB3 and recloned into a
pBD64-based vehicle (Minton et al., 1990), yielding
pMJ51 (Fig. la).
(b) Immunoblot visualization of SubC and precursors
The subC gene encodes a preprosubtilisin (prepro-
SubC). The pre- and pro-peptides are sequentially
removed during translocation from the cell, with release
of mature SubC to the medium.
Immunoblotting of culture fractions of wt Bs strain
168 expressing subC from either pMJ51 (not shown) or
pMJ57 (section f, Fig. 2) revealed specific polypeptides of
the sizes expected of the cell-associated primary precursor
(43 kDa), and the secreted SubC (30 kDa). When these
strains were grown at 30C, a 38-kDa protein was also
observed in the medium early in stationary phase (Fig. 2).
This protein was diminished, and absent in samples gen-
DraI ~771 StuI 240
TATTAATAAC C ~ T T T TAAATTGGTC CTCCAAAAAA ATAGGCCTAC CATATAATTC
I ~ 300
ATTTTTTTTC
-i0 **f | DraI
TATAATAAAT TAACAGAATA ATTGGAATAG ATTATATTAT CCTTCTATTT
II
AAATTATTCT ~ A A T A A A G A G GAGGAGAGTG AGTA
RBS
Fig. 1. Chromosomal m a p and sequence of the 5' region of Bl 6816
subC. (a) subC region in 6816 chromosome, and in clones described in
sections a, h, d and f (not to scale). The extent of the subC fragment
used as a hybridization probe in Southern blotting is also indicated.
Black box, subC gene; S, Sau3Al; T, TaqI; numbers refer to sequence
in panel h. Distance between Bell sites is 2.4 kb. Aval, Sacll and BclI
(5') sites lie about 200, 600 and 1600 bp from the subC start codon. (h)
subC 5' U T R region, non-coding strand. This sequence has GenBank
accession No. X03341. Asterisks indicate tsp; RBS, underlined; - 1 0
homologies, double underlined; homology to aprE D e g U / Q target,
boxed; restriction sites, bold type; palindromes, double-ended arrows.
AG values for palindrome I were - 14.8, or 12.4 kcal, for 1I 5.8, or
- 7 . 2 kcal, calculated as, respectively, described by Tinoco et al. (1973)
and Turner et al. (1988). Numbered open arrows above the sequence
indicate deletion endpoints 769, 770 and 771. Methods: Sequences were
obtained on both strands by enzymatic methods (Messing et al., 1981)
except for sequence 5' from ArM, which was determined only in the
Y-5' direction by chemical methods ( M a x a m and Gilbert, 1977), with
end-labelling of a ClaI linker inserted into the Stul site. Sequences
shown were u n a m b i g u o u s on repeated gels.
erated at 37C and 42C, respectively (not shown). Such
transient appearance of a soluble species the size of par-
tially processed pro-SubC has previously been reported,
and a product-precursor relationship with the mature
30-kDa SubC protein inferred (Schulein et al., 1991).
No corresponding soluble pro-AprE species has been
visualized. Soluble pro-forms were observed with SubC-
protein G fusions only when a defective pro-region hin-
dered proper maturation (Egnell and Flock, 1992).
Moreover, a kinetic analysis of pre-pro-SubC fusion pro-
tein secretion demonstrated that proform was exclusively
cell-associated, and crucially, that mature processed
species remained cell-associated for a period prior to
release to the medium (Jacobs et al., 1993). Therefore,

1 2 3 4 5 compared in all three cloned genes, subC 641 differed in
kDa kDa
66 _
45--
38
29-- 30
Fig. 2. Immunoblot visualization of SubC synthesized by Bs
168[pMJ57]. Lanes: 1, T 2 cell fraction (0.2% xylose); 2-5, medium
fraction (0.05% xylose),To, TI, Tz, T3.5, respectively.Cell and medium
samples corresponded to 20 lal and 150 p.l culture, respectively. The
position of size standards and the various SubC species (open arrows)
is indicated. Methods: ACm rpMJ57 (section f) transformant of Bs 168
trpC2 (BGSC 1A1) was grown at 30'~Cin LB medium, containing 6 lag
Cm/ml, with indicated concentrations of xylose, l-ml samples were har-
vested at indicated times. Sample preparation, separation in 0.1%
SDS-10% PA gels and Western blotting were performed as described
(Jacobs et al., 1993), but with trichloracetic acid precipitation of cells
prior to lysozyme treatment. 1 mM phenylmethylsulfonylfluoridewas
present in all except gel buffers. Anti-SubC serum was pre-adsorbed
with Bs 168 extracts, and was used in the presence of 1% skim milk.
Exoprotease activity of culture medium dilutions was assayed in 0.1 M
Na.phosphate buffer pH 8.0 using 20 lag substrate/ml (DelMar et al.,
1979). Activity units were defined as the change in A4t 2 1000/rain.
The activity of commercial SubC (Sigma, St. Louis, MO, USA) (12-35
units/lag) served to estimate secreted SubC; estimates agreed well with
immunoblot signals (not shown).
the observed soluble SubC 38-kDa material most proba-
bly comprises molecules that have escaped the normal
maturation route.
(c) Conservation of the 5' subC sequence
The region upstream from subC 6816 was sequenced
(Fig. lb). Two other subC sequences have been reported:
subC 2709 (Y. Hong, Chinese Academy, Beijing, personal
communication) and subC 641 ( G a m o n et al., 1989).
These genes were obtained as Sau3AI clones (from site
at nt 36, Fig. lb) and are almost identical, but differ sig-
nificantly from subC 6816.
The 5' part of subC, including the promoter and some
regulatory sequences (see sections d and g) appears to be
especially well conserved. With but a single exception (nt
42 is A in subC 2709), subC 641, 2709 and 6816 are iden-
tical through the available 5' U T R , and coding regions
as far as codon + 101 (numbered as in Jacobs et al., 1985).
This sequence comprises 911 bp, including 206 codons.
Variation in the remaining 3' subC region is higher: the
last 173 codons in subC 2709 and 641 differ from subC
6816 in 23 positions (22 codons, 3 non-conservative alter-
ations). Of 87 bp in the immediate 3' U T R that could be
71
the same five positions from 6816, and 2709. In a further
30 bp, there were three differences between 6816 and
2709. The transcriptional terminator palindrome (t, sec-
tion e) was identical in all three cases.
(d) The subC tsp in BI and Bs
The tsp of subC m R N A isolated from stationary phase
Bl cells at T12 (12 h after cessation of exponential growth)
was determined from a synthesized complementary
strand (cDNA) (Fig. 3A). At a distance of 2 nt from the
largest c D N A sequencing product (lane 1) was an intense
band representing unreacted full-length cDNA. This
resolved into two equally intense signals on briefer expo-
sure (lane 6). This apparent heterogeneity in the size of
the full-length c D N A strand presumably reflects that tsp
can be either at A 262 o r A 263, as indicated in Fig. lb.
A similar 'full-length' doublet was observed when the
tsp of subC m R N A extracted from stationary phase
Bs[pMJ51 ] was mapped (not shown).
At an appropriate distance from the tsp (nt 251 256,
Fig. lb), and also 18 bp further upstream, are sequences
consistent with the consensus - 10 region (TATAAT) for
the major Bs sigma A factor. No convincing match with
the corresponding - 3 5 region consensus (TTGACA)
was found (Moran et al., 1982). The tsp assignation is
consistent with available subC expression data, account-
ing for the absence of subC expression in pSUB3 (Fig. la;
section a), as well as the promoter activity of deletion 771
(Fig. lb; section g). The Bs aprE tsp lies similary close to
the aprE start codon (Ferrari et al., 1988).
(e) The subC transcriptional termination site
Mapping the 3' end of subC m R N A (Fig. 3B) to the
distal end of a palindrome found 3' from the subC stop
codon (nt 1271 1305; Jacobs et al., 1985) defined the
length of native subC transcripts (1269 nt). The subC t
has a G + C - r i c h stem, but lacks the uninterrupted run
of T residues at the distal end typical of Rho-independent
E. coli t (Rosenberg and Court, 1979), and other mapped
Bacillus t (see, for example, Kallio, 1986).
(f) Optimization of subC expression in Bs
Striking features of the subC 5'-UTR, not found in the
homologous aprE, are two overlapping palindromic
sequences (I,II, Fig. lb) whose extent, location 3' from
the tsp and proximity to the RBS, suggest a possible func-
tional significance. Competing m R N A conformations
resulting from predicted folding of I or II might affect
SubC production. The effect of palindrome deletions on
SubC production in Bs 168 was tested in plasmid con-
structs. Promoterless subC DNA, with either StuI, or
DraI at its 5' end, was recloned to a pBD64-based expres-
72
sion vector, pSX50 (Hastrup, 1987), yielding, respectively,
pMJ29 (retaining palindromes) and pMJ57 (lacking pal-
indromes) (Fig. 1). In both plasmids the xylosidase opera-
tor-promoter, O/P~y,B, permitted xylose-regulated subC
transcription. After 24 h growth in MSM with 0.2%
A B 1 2 3 4
123 4 5 6 mRNA half-lives were not measured, it is likely that
!ii~i!i~i~i!i
ii!ii
!ii
!!iii
4112
i nt
Fig. 3. Mapping Bl subC mRNA. (A) Mapping the tsp. Autoradiogram
of sequencing reaction products of cDNA strand synthesized on subC
mRNA template by primer extension. Lanes: 1, cDNA A + G reaction;
2-5, fragment X, respectively, G, A + G , T + C , C reactions. The thin
arrow (lane 1) indicates the last G that can be read off the cDNA, and
corresponded to C at nt 264 in Fig. lb. Lane 6 shows lane 1 after brief
exposure and at higher magnification. The open arrowhead indicates
the longer full-length primed product. Methods: Bl RNA was isolated
(Gilman and Chamberlin, 1983) at T~, and T12 from 20-ml culture grown
at 37'C in modified Schaeffer's medium (MSM; Leighton and Doi,
1971), yielding 500 gg RNA. Northern blotting (1% agarose-2.2 M
formaldehyde gels, nick-translation labelling of the 530-bp Stul-HindIIl
subC DNA probe, hybridization in 50% formamide, autoradiography)
was performed (Maniatis et al., 1982) with Gene Screen membranes.
No signal was obtained at T6. T12 RNA thus served as template for
primed synthesis of a 5' end-labelled DNA strand complementary to
the 5' end of subC mRNA (cDNA). Kinasing of 50 pmol gel-purified
16-mer primer (complementary to subC codons - 9 2 to 97), reverse
transcriptase incubation with 30 gg RNA in the presence of RNasin,
and product separation on a 7 M urea-10% denaturing PA gel werc
performed as described (Maniatis et al., 1982). The labelled cDNA
revealed by autoradiography was excised, subjected to the A + G -
specific sequencing reaction (Maxam and Gilbert, 1977), and separated
as before with sequencing products of another, unrelated fragment as
size markers, followed by autoradiography. (B) subC mRNA 3' end
xylose, Bs[pMJ57] typically accumulated 0 . 5 - l i n g
SubC/ml, compared with about 0.1 mg/ml for
Bs[pMJ29]. Under the same conditions (but without
xylose) Bs [ pMJ 51 ] yielded only 2-14 lag/ml.
Steady-state levels of pMJ29- and pMJ57-supported
subC mRNA, quantified in mid-log and stationary phase
cultures by primer-extension (see legend to Fig. 3A),
reflected this differential SubC production. Although
native B1, and pMJ29-based transcripts, which both
include palindrome I, are comparatively unstable.
Energetically favoured secondary structure could be a
target for endonuclease attack at the 5' extremity of subC
mRNA, and may account for the observed low SubC
production in Bl 6816 and Bs[pMJ51 ] under experimen-
tal conditions. The palindromes possibly serve some reg-
ulatory purpose in B1.
(g) Localization of DegU, DegQ target sites 5' from subC
To test whether the cloned PsubC included sites for regu-
latory proteins, a subC::lacZ translational fusion with
deletions 5' from Ps,bc was generated, and expression of
a chromosomally-integrated copy of each deletion vari-
ant was examined in wt and regulatory mutants of Bs
168 (Fig. 4).
The subC::lacZ fusion expression increased rapidly
immediately after T o (Fig. 4a), mimicking aprE expres-
sion (Ferrari et al., 1988). However, this was unexpected
because Ps,bc-dependent exoproteolytic activity was
detectable in Bl and Bs only several hours after T o
(M.F.J., unpublished observations), and subC RNA was
not detected in Northern blots of BI 6816 T6 RNA (see
legend to Fig. 3A). These negative results may be due to
the low levels of SubC produced in wt strains under
experimental conditions. Fig. 4a also illustrates that in
the wt 168 background, promoter deletions 770 and 771
(having endpoints at, respectively, - 1 0 8 and - 4 8 from
tsp) altered neither the temporal regulation nor the level
of expression of the subC::lacZ fusion gene. This is consis-
tent with the lack of effect on aprE expression observed
in the wt, except when promoter deletion endpoints were
very close to tsp (Ferrari et al., 1988; Henner et al., 1988a).
mapping. Autoradiograph of S1 digestion products on 40-cm 7 M
urea-8% denaturing PA gel. Lanes: 1, 152-bp probe; 2, S1 digest of
annealed Bl T12 RNA and probe; 3 and 4, respectively, A + G and T + C
sequencing reaction products. The arrow indicates the fragment pro-
tected from digestion by S1. Methods: Bl mRNA was subjected to S1
nuclease (Boehringer-Mannheim, Indianapolis, IN, USA) digestion
(Gilman and Chamberlin, 1983) after annealing to a 152-bp Bcll-BamHl
fragment of pSUB3, which overlapped the suhC 3' end by 8 codons
(Jacobs et al., 1985). Probe was labelled at the Bcll end using Pollk
and [~-32P] dNTP.
 73

12
30 C

i oto10 . // 201 o

lo

, o ~ = , - , ,

-2 -1 0 1 2 3 4 -2 -1 0 1 2 3 4 -3 -2 -1 0 1 2 3 4
Time (h) Time (h) Time (h)
Fig. 4. Effect of regulatory mutations on subC::lacZ expression. J3Gal activity in subC::lacZ integrants of Bs 168 (a), Bs degU32(Hy) (b), and Bs
degQ36 (Hy) (e). Solid squares, Bs 168 subC::lacZ 769; solid circles, subC::lacZ 769, solid triangles, subC::lacZ 770; open squares, subC::lacZ 771.
Zero h refers to To. Note different scales on y axis. Methods" To construct a subC::lacZ fusion, subC was subjected to BAL 31 digestion starting with
HindIII-linearized pMJ51 (Fig. 1). pMJ51 has a vector-derived EcoRI site just 5' from AvaI. After Pollk end-repair and ligation with an excess of
HindIII linkers (5'-CAAGCTTG), EcoRI-HindIII promoter fragments (200 300 bp) were recovered on gels from the pooled cloned plasmids extracted
from Bs transformants. These were recloned into corresponding sites 5' from 'lacZ in a pNM480 derivative (Minton, 1984). Plasmids from Ap~Lac +
E. coli transformants selected on agar containing 5-bromo-4-chloro-3-indolyl-[3-D-galactopyranoside were sequenced using ' - 4 0 ' primer (New England
Biolabs, #1212, Beverly, MA, USA). In one, pMJ552, the subC::lacZ junction was in the 9th subC codon. For generation of deletions 5' from Ps,bC,
subC::lacZ was first recloned to a pBR322 derivative having about 300 bp of unrelated DNA between the fusion, and the Ap R gene, yielding pMJ562A.
BAL 31 deletions into the promoter region were then generated from pMJ562A linearized at the (unique) AvaI-adjacent EcoRI site as described
above. After ligation with excess EcoRl linkers (5'-GGAATTCC), the extent of deletion in EcoRI-HindIII Ps,bc fragments of plasmid from 30 Ap R
Lac + E. coli transformants was estimated on 1.7% agarose gels. Among transformants, 29 had little or no deletion, or else ended close to the StuI
site. A single deletion ended midway between these groups. From a representative of each group, a 3-kb EcoRI-SacI fragment carrying P~,hcsubC'::'lacZ'
was recloned into corresponding sites of integration vector pAF1 (Fouet and Sonenshein, 1990), reconstituting the lacZ moiety. Resulting pAF1
derivatives were pMJ769, 770, and 771. Deletion endpoints/site of linker integration (Fig. lb) were established by sequencing as above. Single copy
integrants of the subC::lacZ fusions at the Bs amyE locus were obtained by Bs 168 trpC2 (BGSC 1A1 ) competent cell transformation with either gel-
purified PstI-linearized plasmid pMJ769, pMJ770, or 771, selection for Cm R, followed by purifications in the absence of Cm. Dilutions of chromosomal
DNA from these integrants were used to transform Bs aid-1 degQ36 (Hy) (BGSC 1A53) and Bs trpC2 hprlO (BGSC IA178) competent cells to Cm R.
Competent cells of wt integrants were transformed with chromosomal DNA of QB 4371 trpC2 degS + degU32(Hy)::aphA3 (T. Msadek, personal
communication), with selection for the degSU-linked kanamycin-resistance-encoding gene. For ~Gal activity determination, overnight LB starter
cultures were grown at 37"C from a fresh colony of Bs subC::lacZ integrants, and 100ml prewarmed LB inoculated from these to A45o=0.05.
Incubations were in baffled flasks with vigorous aeration. The wt 168::subC-lacZ 769 strain was always included as an internal control. The growth
kinetics of the three strains were identical, plateauing in different experiments at A45o 1.2 1.6 (not shown). At intervals, 2 2 ml of culture were
withdrawn, centrifuged at 5000 g for 3 min at 4:'C, and cell pellets stored at - 2 0 C for assay. [3Gal activity, expressed as (mean) Miller units, was
determined in duplicate (Miller, 1972), except that solubilization was by lysozyme (100 ~tg lysozyme/ml in Z buffer, 5 min at 37C). Bs 168 J3Gal
activities before and after integration of fusion 769 were, respectively, 10 and 950-2700 units, measured at T2. 5.

The effects of regulators such as DegQ, DegU and Hpr,
which act at sites 5' from the aprE promoter, are thus
not apparent in a wt background. Overproduction of the
positive regulators DegQ or DegU (Yang et al., 1986;
Henner et al., 1988b), or lack of negative regulator protein
Hpr (Perego and Hoch, 1988) result in DegQ(Hy),
DegU(Hy) and Hpr phenotypes, respectively, all of which
are associated with increased aprE transcription (Henner
et al., 1988a; Perego and Hoch, 1988).
The effects of degQ36(Hy), degU32(Hy) and hprlO
mutations on subC::lacZ J3Gal activity were tested. The
degU32(Hy) and degQ36(Hy) mutations stimulated the
expression of subC::lacZ 769 40-and 20-fold, respectively,
compared with the wt background. In contrast, no detect-
able stimulation was observed with fusions subC::lacZ
770 and 771 (Fig. 4b,c), suggesting that targets for activa-
tors DegU and DegQ lie between the endpoints of fusions
769 and 770, a region that is G + C - r i c h (Fig. lb).
Whether this region alone would suffice for subC stimula-
tion in degU and degQ (Hy) mutants seems unlikely, for
reasons explained below.
A consensus sequence, A M T G A A M A K W W (where
K = G or T, M = A or C and W = A or T)(Fig. lb, boxed
sequence), can be found in the 5'-UTR of subC, and Bs
aprE and levansucrase-encoding sacB genes, all of which
are regulated by DegU and DegQ. In aprE, the consensus
(between nt - 1 6 1 and - 1 5 1 relative to tsp) lies in a
region crucial for stimulation by degQ and degU (Hy)
mutations; the aprE 5'-UTR deleted to - 164 allowed full
stimulation, most of which was lost upon deletion to
- 141 (Henner et al., 1988a). The subC homology to the
consensus/aprE DegU/Q target lies within subC':lacZ
770, which was not stimulated at all by the degQ and
degU (Hy) mutations, but the similarities suggest that it
probably comprises a part of the DegU/Q target in Bl.
The lack of subC::lacZ stimulation observed in hprlO
74
(not s h o w n ) was n o t u n e x p e c t e d . F o r aprE s t i m u l a t i o n ,
sites of c o n t a c t b e t w e e n H p r a n d aprE 5 ' - U T R lying
a b o u t 300 bp 5' f r o m tsp w e r e i n d i s p e n s i b l e ( S t r a u c h a n d
H o c h , 1993). A s s u m i n g t h a t Bl has a n H p r h o m o l o g u e
t h a t f u n c t i o n s in a s i m i l a r fashion, its a c t i o n c o u l d n o t
h a v e b e e n o b s e r v e d , since f u s i o n 769 e x t e n d e d o n l y as
far as nt - 1 5 4 f r o m subC tsp.
(h) C o n c l u s i o n s
(1) T h e r e m a r k a b l e d e g r e e of c o n s e r v a t i o n f o u n d in the
5'-UTR', the 5' c o d i n g region, a n d the t of subC genes of
different p r o v e n a n c e , p r e s u m a b l y reflects e v o l u t i o n a r y
c o n s t r a i n t s t h a t d o n o t o p e r a t e o n the rest of the g e n e
and surrounding sequences.
(2) T h e subC gene, e x p r e s s e d in Bs, w a s r e g u l a t e d in a
s i m i l a r f a s h i o n to t h e h o s t ' s h o m o l o g o u s aprE gene.
(3) In c o n t r a s t to the Bs aprE gene, the D e g U / Q t a r g e t
site in Bl subC e x t e n d s in t h e o p p o s i t e (5') d i r e c t i o n f r o m
a r e g i o n c o n s e r v e d in b o t h genes. W h e t h e r t h e c o n s e r v e d
s e q u e n c e is r e q u i r e d for t h e a c t i o n of D e g U / Q o n subC
is n o t clear.
(4) T h e tsp a n d t signals for Bl subC h a v e b e e n defined.
ACKNOWLEDGEMENTS
I w i s h to t h a n k T h e a G a e r t n e r , K i r s t e n H a n s e n , A n n e -
M a r i e B u n d g a a r d a n d B e t h P a r s o n s for a b l e a n d e n t h u s i -
astic a s s i s t a n c e at different times, the D T H AMG 1991
class for g e n e r a t i n g B A L 31 d e l e t i o n s , a n d the C h a i r m a n
of the M i c r o b i o l o g y D e p a r t m e n t for t h e p r o v i s i o n of
e x c e l l e n t facilities.
REFERENCES
Amory, A., Kunst, F., Aubert, E., Klier, A. and Rapoport, G.:
Characterization of the sacQ genes from Bacillus lichen([brmis and
Bacillus subtilis. J. Bacteriol. 169 (1987) 324 333.
DelMar, E.G., Largman, C., Brodrick, J.W. and Geokas, M.C.: A sensi-
tive new substrate for chymotrypsin. Anal. Biochem. 99 (1979)
316 320.
Dubnau, E., Weir, J., Nair, G., Carter 1II, L., Moran Jr., C. and Smith,
I.: Bacillus sporulation gene spoOH codes for sigma 3 (sigmaU).
J. Bacteriol. 170 (1988) 1054 1062.
Egnell, P. and Flock, J.-I.: The autocatalytic processing of the subtilisin
Carlsberg pro-region is independent of the primary structure of the
cleavage site. Mol. Microbiol. 6 (1992) 1115-1119.
Ferrari, E., Henner, D.J., Perego, M. and Hoch, J.A.: Transcription of
Bacillus subtilis subtilisin and expression of subtilisin in sporulation
mutants. J. Bacteriol. 170 (1988) 289 295.
Fouet, A. and Sonenshein, A.L.: A target for carbon source-dependent
negative regulation of the citB promoter of Bacillus subtilis.
J. Bacteriol. 172 (1990) 835-844.
Gamon, K., Berger, H., H/inggl, U., Markgraf, M. and Kreft, J.:
Hybridplasmide zur Erzuegung yon Subtilisin Carlsberg in Bacillus.
European Patent 0348814~ 22nd June, 1989.
Gilman, M.Z. and Chamberlin, M.J.: Development and genetic regula-
tion of B. subtilis genes transcribed by sigma-28 RNA polymerase.
Cell 35 (1983) 285 293.
Hastrup, S.: Gene expression system. European Patent 0242220B1,
21st July, 1993.
Henner, D.J., Eerrari, E., Perego, M. and Hoch, J.A.: Location of the
targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in
upstream regions of the subtilisin promoter. J. Bacteriol. 170
(1988a) 296 300.
Henner, D.J., Yang, M. and Ferrari, E.: Localization of Bacillus subtilis
sacU(Hy) mutations to two linked genes with similarities to the
conserved procaryotic family of two-component signalling systems.
J. Bacteriol. 170 (1988b)5102 5109.
Jacobs, M., Eliasson, M., Uhlen, M. and Flock, J.l.: Cloning, sequencing
and expression of subtilisin Carlsberg from Bacillus lichen!fbrmis.
Nucleic Acids Res. 13 (1985) 8913 8926.
Jacobs, M.F., Andersen, J.B., Kontinen, V. and Sarvas, M.: Bacillus
subtilis PrsA is required in vivo as extracytoplasmic chaperone for
secretion of active enzymes synthesized either with or without pro-
sequences. Mol. Microbiol. 8 (1993)957 966.
Kallio, P.: The effect of the inverted repeat structure on the production
of the cloned B. amyloliquqlaciens a-amylase. Eur. J. Biochem. 158
(1986) 491 495.
Leighton, T.J. and Doi, R.H.: The stability of messenger ribonucleic
acid during sporulation in Bacillus subtilis. J. Biol. Chem. 246 ( 1971 )
3189 3195.
Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, 1982.
Maxam, A.M. and Gilbert, W.: A new method for sequencing DNA.
Proc. Natl. Acad. Sci. USA 74 (1977) 560 564.
Messing, J., Crea, R. and Seeburg, P.H.: A system for shotgun DNA
sequencing. Nucleic Acids Res. 9 (1981) 309 321.
Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1972.
Minton, N.: Improved plasmid for the isolation of translational lac gene
fusions. Gene 31 (1984) 269 273.
Minton, N.P., Swinfield, T.-J., Brehm, J.K. and Oultram, J.D.: The
Gram-postive cloning vector pBD64 arose by a 1844 bp deletion of
pC194 derived DNA. Nucleic Acids Res. 18 (1990) 1651.
Moran, C.P., Lang, N., LeGrice, S.F.J., Lee, G., Stephens, M.,
Sonenshein, A., Pero, J. and Losick, R.: Nucleotide sequences that
signal the initiation of transcription and translation in Bacillus subti-
lis. Mol. Gen. Genet. 186 (1982) 339 346.
Perego, M. and Hoch, J.A.: Sequence analysis and regulation of the hpr
locus, a regulatory gene for protease production and sporulation in
Bacillus subtilis. J. Bacteriol. 170 (1988) 2560 2567.
Rosenberg, M. and Court, D.: Regulatory sequences involved in the
promotion and termination of RNA transcription. Annu. Rev.
Genet. 13 (1979) 319 353.
Schulein, R., Kreft, J., Gonski, S, and Goebel, W.: Preprosubtilisin
Carlsberg processing and secretion is blocked after deletion of amino
acids 97 101 in the mature part of the enzyme. Mol. Gen. Genet.
227(1991) 137 143.
Strauch, M.A. and Hoch, J,A.: Transition-state regulators: sentinels of
Bacillus subtilis post-exponential gene expression. Mol. Microbiol.
7(1993) 337 342.
Tinoco Jr., 1., Borer, P.N., Dengler, B., Levine, M.D., Uhlenbeck, O.C.,
Crothers, D.M. and Gralla, J.: Improved estimation of secondary
structure in ribonucleic acids. Nature New Biol. 246 (1973) 40 41.
Turner, D.H., Sugimoto, N. and Freier, S.M.: RNA structure prediction.
Annu. Rev. Biophys. Biophys. Chem. 17 (1988) 167- 192.
Yang, M., Ferrari, E., Chen, E. and Henner, D.J.: ldentitication of the
pleiotropic sacQ gene of Bacillus subtilis. J. Bacteriol. 166 (1986)
113 119.