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

Correspondence to: Dr. M. F. Jacobs at her present address: Department !3Gal; LB, Luria-Bertani medium (1% Bacto-tryptone/0.5% yeast
of Microbiology, Building 231, University of Maryland, College Park, extract/0.5% NaC1 pH 7.5); MSM, modified Schaeffer's medium; nt,
MD 20742, USA Tel. (1-301) 405-5430; Fax (1-301) 314-9489. nucleotide(s); O, operator; p, plasmid; P, promoter; PA, polyacrylamide;
PolIk, Klenow (large) fragment of E. coli DNA poIymerase I; R, resis-
Abbreviations: aa, amino acid(s); A45o, absorbance at 450 nm (1 cm); tant/resistance; RBS, ribosome-binding site(s); sacB, levansucrase-
Ap, ampicillin; aphA3, kanamycin-encoding gene; aprE, alkaline serine encoding gene; SDS, sodium dodecyl sulfate; subC, subtilisin Carlsberg
protease (subtilisin)-encoding gene of B. subtilis; B., Bacillus; [3Gal, gene; t, transcription terminator(s); To, time end of exponential growth;
13-galactosidase; BGSC, Bacillus Genetic Stock Center; Bl, Bacillus T1,2, etc., hours after To; tsp, transcription start point(s); UTR,
licheniformis; bp, base pair(s); Bs, Bacillus subtilis; cDNA, complemen- untranslated region(s); xynB, xylosidase-encoding gene; wt, wild type;
tary DNA; Cm, chloramphenicol; AG, Gibbs free energy; kb, kilobase(s) [ ], denotes plasmid-carrier state; ::, novel junction (fusion or insertion);
or 1000 bp; Lac + , phenotypically expressing lacZ; lacZ, gene encoding ' (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, DraI ~771 StuI 240
TATTAATAAC C ~ T T T TAAATTGGTC CTCCAAAAAA ATAGGCCTAC CATATAATTC
Fig. la) has previously been described (Jacobs et al., I ~ 300
1985), but it did not display endogenous promoter activ- ATTTTTTTTC
-i0 **f | DraI
TATAATAAAT TAACAGAATA ATTGGAATAG ATTATATTAT CCTTCTATTT
II
ity. To clone the promoter and regulatory sequences
required in cis, restriction sites 5' from subC in 6816 chro- AAATTATTCT ~ A A T A A A G A G GAGGAGAGTG AGTA
RBS
mosomal DNA were first mapped by Southern blotting
analysis. Relevant sites are shown in Fig. la. 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
Attempts to clone 5'-UTR on a 2.4-kb BclI Bl DNA
sections a, h, d and f (not to scale). The extent of the subC fragment
fragment (Fig. la) into either E. coli or Bs hosts using a used as a hybridization probe in Southern blotting is also indicated.
variety of cloning strategies failed; subC-specific clones Black box, subC gene; S, Sau3Al; T, TaqI; numbers refer to sequence
all had deletions or rearrangements at the 5' end. One 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)
such plasmid clone, pMJ2, obtained in low copy in E.
subC 5' U T R region, non-coding strand. This sequence has GenBank
coli, was characterized further. accession No. X03341. Asterisks indicate tsp; RBS, underlined; - 1 0
Southern blotting comparison of pMJ2 with 6816 homologies, double underlined; homology to aprE D e g U / Q target,
chromosomal DNA revealed that the 5' limit of contigu- 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
ous Bl subC sequences lay between the closely spaced
- 7 . 2 kcal, calculated as, respectively, described by Tinoco et al. (1973)
TaqI sites, and the SacI! site (Fig. la,b). Expression and Turner et al. (1988). Numbered open arrows above the sequence
studies showed that the native subC promoter (P~,bC), as indicate deletion endpoints 769, 770 and 771. Methods: Sequences were
well as regions required for regulated subC expression obtained on both strands by enzymatic methods (Messing et al., 1981)
except for sequence 5' from ArM, which was determined only in the
(see sections b and g) were present in pMJ2, despite the
Y-5' direction by chemical methods ( M a x a m and Gilbert, 1977), with
rearrangement. The subC gene was reconstituted from end-labelling of a ClaI linker inserted into the Stul site. Sequences
material in pMJ2 and pSUB3 and recloned into a shown were u n a m b i g u o u s on repeated gels.
pBD64-based vehicle (Minton et al., 1990), yielding
pMJ51 (Fig. la).
erated at 37C and 42C, respectively (not shown). Such
(b) Immunoblot visualization of SubC and precursors transient appearance of a soluble species the size of par-
The subC gene encodes a preprosubtilisin (prepro- tially processed pro-SubC has previously been reported,
SubC). The pre- and pro-peptides are sequentially and a product-precursor relationship with the mature
removed during translocation from the cell, with release 30-kDa SubC protein inferred (Schulein et al., 1991).
of mature SubC to the medium. No corresponding soluble pro-AprE species has been
Immunoblotting of culture fractions of wt Bs strain visualized. Soluble pro-forms were observed with SubC-
168 expressing subC from either pMJ51 (not shown) or protein G fusions only when a defective pro-region hin-
pMJ57 (section f, Fig. 2) revealed specific polypeptides of dered proper maturation (Egnell and Flock, 1992).
the sizes expected of the cell-associated primary precursor Moreover, a kinetic analysis of pre-pro-SubC fusion pro-
(43 kDa), and the secreted SubC (30 kDa). When these tein secretion demonstrated that proform was exclusively
strains were grown at 30C, a 38-kDa protein was also cell-associated, and crucially, that mature processed
observed in the medium early in stationary phase (Fig. 2). species remained cell-associated for a period prior to
This protein was diminished, and absent in samples gen- release to the medium (Jacobs et al., 1993). Therefore,
71

1 2 3 4 5 compared in all three cloned genes, subC 641 differed in


the same five positions from 6816, and 2709. In a further
kDa kDa 30 bp, there were three differences between 6816 and
66 _ 2709. The transcriptional terminator palindrome (t, sec-
tion e) was identical in all three cases.
45--
38 (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)
29-- 30 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
Fig. 2. Immunoblot visualization of SubC synthesized by Bs band representing unreacted full-length cDNA. This
168[pMJ57]. Lanes: 1, T 2 cell fraction (0.2% xylose); 2-5, medium resolved into two equally intense signals on briefer expo-
fraction (0.05% xylose),To, TI, Tz, T3.5, respectively.Cell and medium
sure (lane 6). This apparent heterogeneity in the size of
samples corresponded to 20 lal and 150 p.l culture, respectively. The
position of size standards and the various SubC species (open arrows) the full-length c D N A strand presumably reflects that tsp
is indicated. Methods: ACm rpMJ57 (section f) transformant of Bs 168 can be either at A 262 o r A 263, as indicated in Fig. lb.
trpC2 (BGSC 1A1) was grown at 30'~Cin LB medium, containing 6 lag A similar 'full-length' doublet was observed when the
Cm/ml, with indicated concentrations of xylose, l-ml samples were har-
tsp of subC m R N A extracted from stationary phase
vested at indicated times. Sample preparation, separation in 0.1%
SDS-10% PA gels and Western blotting were performed as described Bs[pMJ51 ] was mapped (not shown).
(Jacobs et al., 1993), but with trichloracetic acid precipitation of cells At an appropriate distance from the tsp (nt 251 256,
prior to lysozyme treatment. 1 mM phenylmethylsulfonylfluoridewas Fig. lb), and also 18 bp further upstream, are sequences
present in all except gel buffers. Anti-SubC serum was pre-adsorbed consistent with the consensus - 10 region (TATAAT) for
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 the major Bs sigma A factor. No convincing match with
Na.phosphate buffer pH 8.0 using 20 lag substrate/ml (DelMar et al., the corresponding - 3 5 region consensus (TTGACA)
1979). Activity units were defined as the change in A4t 2 1000/rain. was found (Moran et al., 1982). The tsp assignation is
The activity of commercial SubC (Sigma, St. Louis, MO, USA) (12-35 consistent with available subC expression data, account-
units/lag) served to estimate secreted SubC; estimates agreed well with
immunoblot signals (not shown). 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 observed soluble SubC 38-kDa material most proba- the aprE start codon (Ferrari et al., 1988).
bly comprises molecules that have escaped the normal
maturation route. (e) The subC transcriptional termination site
Mapping the 3' end of subC m R N A (Fig. 3B) to the
(c) Conservation of the 5' subC sequence distal end of a palindrome found 3' from the subC stop
The region upstream from subC 6816 was sequenced codon (nt 1271 1305; Jacobs et al., 1985) defined the
(Fig. lb). Two other subC sequences have been reported: length of native subC transcripts (1269 nt). The subC t
subC 2709 (Y. Hong, Chinese Academy, Beijing, personal has a G + C - r i c h stem, but lacks the uninterrupted run
communication) and subC 641 ( G a m o n et al., 1989). of T residues at the distal end typical of Rho-independent
These genes were obtained as Sau3AI clones (from site E. coli t (Rosenberg and Court, 1979), and other mapped
at nt 36, Fig. lb) and are almost identical, but differ sig- Bacillus t (see, for example, Kallio, 1986).
nificantly from subC 6816.
The 5' part of subC, including the promoter and some (f) Optimization of subC expression in Bs
regulatory sequences (see sections d and g) appears to be Striking features of the subC 5'-UTR, not found in the
especially well conserved. With but a single exception (nt homologous aprE, are two overlapping palindromic
42 is A in subC 2709), subC 641, 2709 and 6816 are iden- sequences (I,II, Fig. lb) whose extent, location 3' from
tical through the available 5' U T R , and coding regions the tsp and proximity to the RBS, suggest a possible func-
as far as codon + 101 (numbered as in Jacobs et al., 1985). tional significance. Competing m R N A conformations
This sequence comprises 911 bp, including 206 codons. resulting from predicted folding of I or II might affect
Variation in the remaining 3' subC region is higher: the SubC production. The effect of palindrome deletions on
last 173 codons in subC 2709 and 641 differ from subC SubC production in Bs 168 was tested in plasmid con-
6816 in 23 positions (22 codons, 3 non-conservative alter- structs. Promoterless subC DNA, with either StuI, or
ations). Of 87 bp in the immediate 3' U T R that could be DraI at its 5' end, was recloned to a pBD64-based expres-
72

sion vector, pSX50 (Hastrup, 1987), yielding, respectively, xylose, Bs[pMJ57] typically accumulated 0 . 5 - l i n g
pMJ29 (retaining palindromes) and pMJ57 (lacking pal- SubC/ml, compared with about 0.1 mg/ml for
indromes) (Fig. 1). In both plasmids the xylosidase opera- Bs[pMJ29]. Under the same conditions (but without
tor-promoter, O/P~y,B, permitted xylose-regulated subC xylose) Bs [ pMJ 51 ] yielded only 2-14 lag/ml.
transcription. After 24 h growth in MSM with 0.2% 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),

A B 1 2 3 4 reflected this differential SubC production. Although


123 4 5 6 mRNA half-lives were not measured, it is likely that
native B1, and pMJ29-based transcripts, which both
include palindrome I, are comparatively unstable.
!ii~i!i~i~i!i Energetically favoured secondary structure could be a
target for endonuclease attack at the 5' extremity of subC
ii!ii
!ii
!!iii
4112
i nt 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
Fig. 3. Mapping Bl subC mRNA. (A) Mapping the tsp. Autoradiogram
the low levels of SubC produced in wt strains under
of sequencing reaction products of cDNA strand synthesized on subC
mRNA template by primer extension. Lanes: 1, cDNA A + G reaction; experimental conditions. Fig. 4a also illustrates that in
2-5, fragment X, respectively, G, A + G , T + C , C reactions. The thin the wt 168 background, promoter deletions 770 and 771
arrow (lane 1) indicates the last G that can be read off the cDNA, and (having endpoints at, respectively, - 1 0 8 and - 4 8 from
corresponded to C at nt 264 in Fig. lb. Lane 6 shows lane 1 after brief
tsp) altered neither the temporal regulation nor the level
exposure and at higher magnification. The open arrowhead indicates
the longer full-length primed product. Methods: Bl RNA was isolated of expression of the subC::lacZ fusion gene. This is consis-
(Gilman and Chamberlin, 1983) at T~, and T12 from 20-ml culture grown tent with the lack of effect on aprE expression observed
at 37'C in modified Schaeffer's medium (MSM; Leighton and Doi, in the wt, except when promoter deletion endpoints were
1971), yielding 500 gg RNA. Northern blotting (1% agarose-2.2 M very close to tsp (Ferrari et al., 1988; Henner et al., 1988a).
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 mapping. Autoradiograph of S1 digestion products on 40-cm 7 M
the 5' end of subC mRNA (cDNA). Kinasing of 50 pmol gel-purified urea-8% denaturing PA gel. Lanes: 1, 152-bp probe; 2, S1 digest of
16-mer primer (complementary to subC codons - 9 2 to 97), reverse annealed Bl T12 RNA and probe; 3 and 4, respectively, A + G and T + C
transcriptase incubation with 30 gg RNA in the presence of RNasin, sequencing reaction products. The arrow indicates the fragment pro-
and product separation on a 7 M urea-10% denaturing PA gel werc tected from digestion by S1. Methods: Bl mRNA was subjected to S1
performed as described (Maniatis et al., 1982). The labelled cDNA nuclease (Boehringer-Mannheim, Indianapolis, IN, USA) digestion
revealed by autoradiography was excised, subjected to the A + G - (Gilman and Chamberlin, 1983) after annealing to a 152-bp Bcll-BamHl
specific sequencing reaction (Maxam and Gilbert, 1977), and separated fragment of pSUB3, which overlapped the suhC 3' end by 8 codons
as before with sequencing products of another, unrelated fragment as (Jacobs et al., 1985). Probe was labelled at the Bcll end using Pollk
size markers, followed by autoradiography. (B) subC mRNA 3' end 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, Whether this region alone would suffice for subC stimula-
which act at sites 5' from the aprE promoter, are thus tion in degU and degQ (Hy) mutants seems unlikely, for
not apparent in a wt background. Overproduction of the reasons explained below.
positive regulators DegQ or DegU (Yang et al., 1986; A consensus sequence, A M T G A A M A K W W (where
Henner et al., 1988b), or lack of negative regulator protein K = G or T, M = A or C and W = A or T)(Fig. lb, boxed
Hpr (Perego and Hoch, 1988) result in DegQ(Hy), sequence), can be found in the 5'-UTR of subC, and Bs
DegU(Hy) and Hpr phenotypes, respectively, all of which aprE and levansucrase-encoding sacB genes, all of which
are associated with increased aprE transcription (Henner are regulated by DegU and DegQ. In aprE, the consensus
et al., 1988a; Perego and Hoch, 1988). (between nt - 1 6 1 and - 1 5 1 relative to tsp) lies in a
The effects of degQ36(Hy), degU32(Hy) and hprlO region crucial for stimulation by degQ and degU (Hy)
mutations on subC::lacZ J3Gal activity were tested. The mutations; the aprE 5'-UTR deleted to - 164 allowed full
degU32(Hy) and degQ36(Hy) mutations stimulated the stimulation, most of which was lost upon deletion to
expression of subC::lacZ 769 40-and 20-fold, respectively, - 141 (Henner et al., 1988a). The subC homology to the
compared with the wt background. In contrast, no detect- consensus/aprE DegU/Q target lies within subC':lacZ
able stimulation was observed with fusions subC::lacZ 770, which was not stimulated at all by the degQ and
770 and 771 (Fig. 4b,c), suggesting that targets for activa- degU (Hy) mutations, but the similarities suggest that it
tors DegU and DegQ lie between the endpoints of fusions probably comprises a part of the DegU/Q target in Bl.
769 and 770, a region that is G + C - r i c h (Fig. lb). 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 , Gilman, M.Z. and Chamberlin, M.J.: Development and genetic regula-
tion of B. subtilis genes transcribed by sigma-28 RNA polymerase.
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
Cell 35 (1983) 285 293.
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 Hastrup, S.: Gene expression system. European Patent 0242220B1,
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 21st July, 1993.
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 Henner, D.J., Eerrari, E., Perego, M. and Hoch, J.A.: Location of the
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 targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in
upstream regions of the subtilisin promoter. J. Bacteriol. 170
far as nt - 1 5 4 f r o m subC tsp. (1988a) 296 300.
Henner, D.J., Yang, M. and Ferrari, E.: Localization of Bacillus subtilis
(h) C o n c l u s i o n s sacU(Hy) mutations to two linked genes with similarities to the
conserved procaryotic family of two-component signalling systems.
(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
J. Bacteriol. 170 (1988b)5102 5109.
5'-UTR', the 5' c o d i n g region, a n d the t of subC genes of Jacobs, M., Eliasson, M., Uhlen, M. and Flock, J.l.: Cloning, sequencing
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 and expression of subtilisin Carlsberg from Bacillus lichen!fbrmis.
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 Nucleic Acids Res. 13 (1985) 8913 8926.
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