Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus-brevis

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Appl Microbiol Biotechnol (1994) 42:78-84 © Springer-Verlag 1994

M. E. Louw • S. J. Reid • D. M. James T. G. Watson

Cloning and sequencing the deg$.degU operon from an alkalophilic Bacillus brevis

Received: 12 November 1993 / Received revision: 1 February 1994 / Accepted: 21 February 1994

Abstract The sacU region from an alkalophilic Bacil- lus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively.

On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec- strain as well as in a B. subtilis sacU(HY) strain.

introduction

Bacillus subtilis can secrete a wide variety of extracellular enzymes. These include proteases, a-amylase, levansucrase and /3-glucanase (Priest 1977). Many of these enzymes are of industrial importance. Expression of genes encoding extracellular enzymes was found to be under control of the sacU region, which consists of two genes, degS and degU (Henner et al. 1988; Kunst et al.

1988; Tanaka and Kawata 1988).

The genes degS and degU are unlinked to any of the target genes and constitute an operon. The operon en- codes the DegS and DegU proteins, both of which dis- play amino acid sequence homology with the sensor and effector proteins, respectively, of the bacterial two- component regulatory system. In this system one component accepts an environmental signal and transduces the information to the other component, resulting in activation of the target gene or cell machinery (Stock et

M. E. Louw (5:~) • T. G. Watson

Biotechnology Programme, Division of Food Science and Technology, CSIR, P.O. Box 395, Pretoria 0001,

Republic of South Africa. FAX: 27 (12) 841-3651 S. J. Reid • D. M. James

Department of Microbiology, University of Cape Town, Private Bag, Rondebosch 7700, Cape Town,

Republic of South Africa

al. 1989). Similarities were found between DegS and the histidine protein kinase family and between DegU and the response regulator proteins, suggesting that DegS might modify DegU through phosphorylation (Henner et al. 1988; Kunst et al. 1988; Msadek et al.

1990).

The degS and degU genes were initially defined by different classes of mutations leading either to deficiency of degradative enzyme syntheses (designated degS or degU mutations) or to overproduction of degradative enzymes [designated degS(HY) or degU(HY) mutations]. Several of these mutations have been charac- terized at the molecular level (Henner et al. 1988;

Msadek et al. 1990). Both the degS and degU genes were found to be essential for the production of extracellular enzymes in B. subtilis and these gene products were found to exert control over degradative enzyme synthesis at a transcriptional level.

In-vitro phosphorylation experiments using modified DegS and DegU proteins support the hypothesis that degS(HY) and degU(HY) mutations favour accu- mulation of the phosphorylated form of DegU by in- creasing the phosphorylation rate of the response regulator or by enhancing the stability of the phosphorylated protein by decreasing its dephosphorylation rate (Dahl et al. 1991; Tanaka et al. 1991). It was also found that mutations in degS or degU that lead to a deficiency of degradative enzyme synthesis promote the accumu- lation of the unphosphorylated form of DegU. It is in this form that DegU is necessary for competence gene expression (Dahl et al. 1992; Msadek et al. 1991). Thus DegU appears to be the first response regulator shown to have two active conformations controlling two dis- tinct regulatory pathways: degradative enzyme production and expression of genetic competence (Msadek et al. 1993). The in-vivo equilibrium between the two forms of DegU is presumably regulated by DegS in response to an environmental signal that has not, as yet, been identified.

In this report we detail the cloning, nucleotide sequence and characterization of the degS-degU operon

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f r o m B. b r e v i s a n d e v a l u a t e its effect o n e x t r a c e l l u l a r e n z y m e p r o d u c t i o n .

Materials and methods

Strains and plasmids

Transformation

The polyethylene-glycol-induced protoplast transformation pro- cedure of Chang and Cohen (1979) was used with the following modifications: stationary phase recipient cells were diluted 1:50 in Luria broth and grown with shaking to an optical density of 0.35 at 540 nm. Lysozyme treatment was continued for 45 rain and 30% (w/v) polyethylene glycol (Merck approx, relative molecular mass 4000) was used for induction of DNA uptake.

The B. brevis Alk 36 strain was isolated from soil (Louw et al.

1993). All the B. subtilis strains and plasmids were obtained from the Bacillus Genetic Stock Centre and the BGSC accession num- bers are quoted: B. subtilis 1A46 recE4 thr-5 trpC2; B. subtilis 1A311 amyE( + M) arnyR2 met135 pro(L) purF6 sacU9 str; 13. sub- til~s 1A201 hisA1 sacA321 sacU42 trpC2. The plasmid pPL703 is a promoter probe vector containing a promoter-less cat gene. The plasmid pPL708 is an expression vector derived from pPLT03 containing the Spo2 promoter (Mongkolsnk et al. 1983).

Media and qualitative tests

t3. subtilis strains and 13. brevis were grown in Luria broth (10 g tryptone, 5 g yeast extract and 10 g NaC1 per litre) at pH 7.2 and pH 9.0, respectively. All strains were grown at 37 ° C.

DM3 plates were made up as described by Corfield et al.

(1984) and further modified so as to include both 1% soluble starch (Saarchem) and 1% casein (Saarchem) in the plates. Saar- chem soluble starch was used in preference to Merck as it was found to form a more opaque background when incorporated into the DM3 plates together with casein. The volumes were adjusted accordingly to accommodate both the casein and starch.

Chloramphenicol and neomycin were added at concentrations of 10/~g/ml when required, except for DM3 plates where chloramphenicol concentrations of 20/xg/ml were added.

Extracellular enzyme production was determined using the following media: Luria broth plus 1% soluble starch (Merck) for a-amylase determination; Luria broth plus 0.1% lichenan from Cetraria islandica (Sigma) for/3-glucanase determination. For levansucrase production two types of media were used, Luria broth plus 1% sucrose (Amory et al. 1987) and MMCH medium containing 2% sucrose (Kunst et al. 1988). Auxotrophic requirements were added at a concentration of 100 mg/1. The medium for protease production was developed from that of Horikoshi and Ikeda (1977) and contained Bactotryptone, 1%; yeast extract, 0.5%;

NaC1, 1%; soluble starch (Merck), 2%; K2HPO4, 0.1%;

MgSO4"H20, 0.02%. The pHwas adjusted to 7.2 with 1M NaOH.

All enzyme activities were measured after 24 h growth of the strains in the respective media except for levansucrase production where the culture supernatants of exponentially growing cells were used for analysis.

DNA manipulations and cloning procedures

Standard methods were used for chromosomal DNA isolation from B. brevis (Lovett and Keggins 1979) and plasmid DNA isolation (Gryczan et al. 1978), which was purified by caesium chlo- ride-ethidium bromide equilibrium centrifugation according to the method of Sambrook et al. (1989). Partial Sau3A restriction of the B. brevis DNA was carried out and fragments in the size range of 3-6 kb were purified from agarose using the Geneclean kit (Biol01, Calif., USA.)

Restriction enzymes and T4 DNA ligase were from Boehrin- ger Mannheim (Germany) and used according to the manufacturer's specifications.

Nucleotide sequencing

The 13. brevis DNA insert could not be subcloned into Bluescript SK or KS as this proved lethal to the recipient Escherichia coli strain. A similar finding has been reported previously by Kunst et al. (1988). Sequencing templates were therefore generated by subcloning an internal Hind III fragment of 0.4 kb and a series of Sau3A fragments from the B. brevis DNA insert into Bluescript- SK. Primers were then manufactured to complete the sequence in both directions.

The nucleotide sequences of the degS-degU genes were determined by the dideoxy-nucleotide-chain-termination method (San- ger et al. 1977) using the Sequenase II Kit (USB) according to the manufacturer's specifications. The nucleotide and deduced amino acid sequences were analysed using the Genetics Computer Group (GCG) software package (version 7.0). The GenBank/

EMBL accession number is L15444.

Enzyme assays

Protease activity was determined using azocasein as substrate (Millet 1970). Alkaline protease activities were assayed at pH 9.5 in the presence of 20mM ethylenediaminetetraacetic acid (EDTA) and total protease production was measured at pH 7.0.

Activities were expressed in Anson units (Anson 1939) by using protease reference standards of known protease activity, a-Amy- lase was assayed according to the method of Bernfeld (1949). One unit was defined as the number of milligrams of maltose liberated in 3 min at 37 ° C by 1 ml of enzyme solution./~-Glucanase activities were defined as previously described (Louw et al. 1993) and levansucrase was determined according to the method of Kunst et al. (1988). One unit corresponds to 1/xmol glucose produced per minute.

For calculating specific activities the total amount of protein was estimated from the bacterial culture pellets using the biuret method (Herbert et al. 1971).

In-vitro transcription and translation

Covalently dosed circular plasmids were used as templates for the prokaryotic coupled transcription-translation system as rec- ommended by the manufacturer (Amersham Corp., Arlington Heights, Ill., USA). Proteins were labelled with [35S] methionine (specific activity, > 37 TBq/mM) separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 15%

acrylamide to 0.4 bisacrylamide) and visualized by autoradiogra- phy.

Results

C l o n i n g of t h e d e g S - d e g U g e n e s f r o m B. b r e v i s

A g e n o m e l i b r a r y of B. brevis D N A was c o n s t r u c t e d b y l i g a t i n g a p p r o x i m a t e l y 0 . 5 / z g of B. b r e v i s size-frac- t i o n a t e d D N A to 0.5/xg of B a r n H 1 l i n e a r i z e d p P L 7 0 3 a n d t h e l i g a t i o n m i x was t r a n s f o r m e d i n t o B. subtilis 1A46. T h e t r a n s f o r m a n t s w e r e p l a t e d o n t o t h e m o d i f -

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80

Fig. 1 Modified DM3 plate showing two colonies producing haloes, indicating increased protease production

ied DM3 plates. The inclusion of both starch and casein in the DM3 medium was found to facilitate the screening for hyperproducing protease transformants. From approximately 500 colonies, three were identified that gave rise to haloes on the DM3 plates, indicating increased protease production (Fig. 1). These three transformants, designated pML1, pML2 and pML3, as well as B. subtilis 1A46, were inoculated into protease screening medium and grown for 24 h. Protease production was determined at pH 9.5 (Table 1).

When compared to the protease produced by the recipient strain it was found that the production of both neutral and alkaline proteases by the recombinants was substantially increased. A Pstl and EcoR1 double di- gestion of pML1, pML2 and pML3 indicated that all three transformants contained the same 2.6-kb insert.

Thus, the recombinant plasmid with the smallest insert, pML2, was chosen for further study.

Nucleotide sequence determination

The 2.6 kb fragment of pML2 was sequenced in both directions using the dideoxy-chain termination method (Fig. 2). Two complete open-reading frames (ORFs) were identified in the sequence; the degS gene (1133

Table 1 Extracellular protease activities of the three transformants and Bacillus subtilis 1A46 (AU Anson units, EDTA ethylenediaminetetraacetic acid)

Strain Protease activity (~xAU/ml)

With EDTA Without EDTA

pML1 625 1025

pML2 725 1475

pML3 400 675

B. subtilis 1A46 35 47

nt), which has a presumptive GTG start codon at position 126 and ends with an T A G at position 1259, and the degU gene (710 nt), which has a presumptive TTG start codon at position 1294 and ends with a T A A at 2004. These codons initiate translation in a number of Bacillus genes: the B. licheniformis /3-1actamase (Neu- gebauer et al. 1981), B. pumilus cat-86 (Harwood et al.

1983), and B. subtilis alkaline phosphatase genes (Bookstein et al. 1990). The degU gene is preceded by a strong ribosome-binding site RBS, ( 5 ' - G G A G G G - 3 ' ) , eight bases from the TTG start codon. The degS gene has no obvious RBS, but the GTG is followed by GCT, which has been shown to increase initiation at GTG initiation codons (Ringquist et al. 1992). No termination or promoter sequences are located in the 34 bases sep- arating the two ORFs, and no transcription terminator sequences could be identified downstream of the degU gene.

The predicted sizes of the polypeptides encoded by the degS and the degU genes are 378 and 237 amino acids giving calculated molecular masses of 43.8 and 27.0 kDa, respectively.

Sequence comparisons at the nucleotide and amino acid levels indicated that these ORFs show significant similarity to the degS and degU genes of B. subtilis (Henner et al. 1988; Kunst et al. 1988; Tanaka and Ka- wata 1988). The similarity of the deduced amino acid sequences of the B. brevis genes to those of B. subtilis was determined on the basis of identical amino acid sequence comparisons. The alignments revealed that 54% of the amino acids were identical (73% similarity) in the degS genes (Fig. 3), whereas the degU genes have 71% identical amino acids (84% similarity) (Fig. 4).

Effect of the degS-degU operon on the production of extracellular enzymes

The plasmid pML2 containing the degS-degU genes was transformed into the sacU mutant B. subtilis 1A201 and was found to restore levansucrase synthesis in this strain. The degS-degU genes were also transformed into a sacU(HY) mutant strain, B. subtilis 1A311. By using B. subtilis 1A311 (pPL708) and B. subtilis 1A46 (pPL708) as control strains, the effect of the degS-degU operon on extracellular enzyme production could be assessed (Table 2). In all cases the plasmid pML2 carrying the degS-degU genes was found to enhance extracellular enzyme production. It was particularly interest- ing that pML2 was able to enhance the sacU9(HY) mutation found in strain 1A311. This mutation was due to a single nucleotide change that caused a Glu to Lys change at amino acid 107 in ORF2 (Henner et al. 1988).

Despite the fact that B. subtilis 1A46 and B. subtilis 1A311 are not a set of isogenic strains, the effects of the degS-degU operon are sufficiently clear-cut for this not to be a significant factor.

The increases in enzyme levels varied between ten- and 20-fold for total protease production and six- to 36-

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M A D P Q T L D K I I D

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2 4 1 T A G A A C A A G A A C T G C A A G A A G T C C G C A T G A ~ G T T G C A G A G A T T A T A G A C C A A T C G G A C C G G G C G G A A G T A C A C G C T C G A 3 2 0 E Q E L Q E V R M K V A E I I D Q S D R A E V H A R

3 2 1 T T T G C A c G C A A T C G A T T G G C T G A A G T G A G T A A G C A A T T T C A T A G A T A T T c A A A T G A A G A G A T A ~ G T A A A G T G T A C G A G c A 4 0 0

F A R N R L A E V S K Q F H R Y S N E E I R K V Y E Q

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A N E L Q V K L A L L Q Q E E Q Q L R D R R D A I E R

481 G C C G A T T G T T G A A T C T ~ A A A G A T A C C A T C G A G C G T G C G G A A G A A T T A G T T G G G C A G A T G A C c G T T G T c T A C A A ~ T T T C T T 5 6 0

R L L N L K D T I E R A E E L V G Q M T V V Y N F L

5 6 1 A C A G G A G A T c T T C G T C A A G T T G G A G A A G c A C T T G A G G A T G C A A G G G A A A A G C A G G c G T T T G G T C T A c A A A T T A T T C A A G C 640

T G D L R Q V G E A L E D A R E K Q A F G L Q I I Q A

641 T c A ~ A G A G G A A G A A C G T C G A A A G C T T T C c C G G G A A A T T c A T G A ~ G G A C C T G c T C A A A T G A T G G C G A A T G T T C T A T T G C G c T 7 2 0

Q E E E R R K L S R E I H D G P A Q M M A N V L L R S

7 2 1 cAGi~CTGGTTGAGCGCATTTATCACGACA`~GC~ATTGATG/~GCGTTAAAGGA/~TTCGTGATTTC~Gci~C~TGGTG 800

E L V E R I Y H D K I G I D E A L K E I R D L R K M V

8 0 1 A A G T C T T C T C T T G • T G A A G T C C G G C G A A T T A T C T A T G A T T T A • G c C G G A T G G • C c T T G A T G A • C T T G G A C T c A T T c • A A c 8 8 0

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8 8 1 A T T G A A G A A G T A T G T A ~ C C T T T G A A G A A C A T A C G G G A A T A T T T G T T G A T T T T A A A C A C A T A G G A 2 a ~ A G G A G A G C G C T 960

L K K Y V K T F E E H T G I F V D F K H I G K G E R F

961 T C C c A G A G c A T G T G G A G A T C G c A C T A T T T • G C c T T G T T C A G G A A G C G T T A c A A A A C A C A c G A A A G C A T G C A A A A G C T T C A 1 0 4 0

P E H V E I A L F R L V Q E A L Q N T R K H A K A S

1 0 4 1 C A T G T C C A T G T G A A A A T C G A A G A G C A G A A A A C G A A A T T C A C T G T C G T G A T A A ~ G A T A A T G G ~ A A A G G A T T C G A T C A ~ C 1 1 2 0 H V H V K I E E Q K T K F T V V I K D N G K G F D Q T

1 1 2 1 C G A G A A A A A G G A A G G G T C A T T T G G T T T A G T T G G A A T G A A A G A G A G G G T c A A C A T G T T G A A A G G C c A G C T T G T c A T T C G A A 1 2 0 0

E K K E G S F G L V G M ~K E R V N M L K G Q L V I R T

1 2 0 1 c G A A G C C A A A C G A T G G A A C A A C G A T C A T C A T T T C C A T T C C T A T C A c A A C C G A A G A A T A G A C T G C G T A G G T G C A A A A A T T G 1 2 8 0

K P N D G T T I I I S I P I T T E E *

1 2 8 1 G A G G G T G A T T T A A T T G A A C G A G c A A G T A A A C G A A A A c A A A A T T C A A A T T G T C A T C A T T G A T G A C C A T C A A C T A T T C C G T G 1 3 6 0 M N E Q V N E N K I Q I V I I D D H Q L F R E

1 3 6 1 A A G G G G T T A A A C G G A T C C T A G C C A T G G A G C C A G A A T T T G A G G T T G T G G C T G A T G G A G A A G A C G G C G A ~ C G C T G T T G A A 1 4 4 0

G V K R I L A M E P E F E V V A D G E D G E N A V E

1 4 4 1 C T A G T A G A A A A A T A T A A T • C A G A T G T C A T C C T T A T G G A T A T T A A c A T G C C A A A G G T G A A T G G T G T T G A A G c G A c A A G A G A 1 5 2 0

L V E K Y N P D V I L M D I N M P K V N G V E A T R D

1 5 2 1 T T T A A T T c A A A G G T A C C C T G A C G T A A A G G T A T T G G T T c T C T C G A T T C A C G A C G A T G A G T C C T A T G T C A C T c A T G T A C T A A 1 6 0 0

L I Q R Y P D V K V L V L S I H D D E S Y V T H V L K

1 6 0 1 A A A C G G G A G C T T C C G G T T A T C T A T T A A A A G A ~ T G G A C G C C G A C G C T C T A A T T G A A G C A G T A ~ G T C G T A G C G C A A G G C 1 6 8 0

T G A S G Y L L K E M D A D A L I E A V K V V A Q G

1 6 8 1 G G G G C G T A C A T T C A T C C G A A G G T G A C A C A T A A T C T C A T C A A G G A A T A C C G T C G T C T A G T A ~ T G A A G A T G A A C A A G A A A G 1 7 6 0

G A Y I H P K V T H N L I K E Y R R L V N E D E Q E S

1 7 6 1 C T c T G A ~ T C G G C T T T A ~ A A G A A G T c G A A T A T C G c A A G c C G T T G C A C A T T T T A A C T C G T c G T G A A T G T G A A G T G c T A C A G c 1 8 4 0

S E I G F K E V E Y R K P L H I L T R R E C E V L Q L

1 8 4 1 T C A T G A C T G A C G G T T A T A G c A A C C G A A T G A T T G G T G A A G C T C T A T A C A T T A G T G A G A A A A C A G T C A ~ A A A A T C A T G T T T C A 1 9 2 0 M T D G Y S N R M I G E A L Y I S E K T V K N H V S

1 9 2 1 A A T A T T T T G C A A A A G A T G A A T G T A A A C G A T C G A A C A C A A G C G G T A G T A G A A T C G A T C A A ~ C G G T T G G G T A A A A G T A C G 2 0 0 0 N I L Q K M N V N D R T Q A V V E S I K N G W V K V R

2 0 0 1 T T A A T c • A A A A A G • G A T c G c T T C A T T A c G c • c T C T T G A A A G G T C T T T c A A T T A A A G G T A c A G T A A A A A A G A C A A G A T c C G 2 0 8 0

2 0 8 1 G G G A A T T C A C A T C A A T G C G A 2 1 0 0

Fig. 2 Nucleotide sequence of part of the 2.6-kb EcoR1 fragment sequence. The ribosome binding site of the degU gene is under- of pML2 containing the Bacillus brev~ degS and degU genes. The lined and the G T G and T T G start codons are indicated in bo~

nucleotide sequence is numbered throughout. The deduced ami- ~twrs. The T A G ending the degS gene and T A A at the end of the no acid sequence is shown in single-~er code below the coding degU gene are indicated by aswr#ks.

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Fig. 3 Alignment of the deduced amino acid sequence of the B.

brevis (BB) and B. subtilis (BS) degS genes. Identical and conserved amino acids are marked with asterisks and dots, respectively. The conserved histidine-189 residue is boxed and the site of the degS200(HY) mutation in the B. subtilis gene is printed in bold letters.

fold for /3-glucanase production by both B. subtilis strains 1A311 and 1A46 respectively. Levansucrase production increased two- and five-fold when strains 1A46 and 1A311 were grown in minimal medium. It was, however, found that when 1A311 and 1A46 were grown in Luria broth plus sucrose the levansucrase production levels for 1A311 were only increased by 1.3- fold but for 1A46 these levels were increased by 90- fold. When grown in this medium however, 1A46 took twice as long as 1A311 to reach log phase growth. The o~-amylase levels were increased two-fold in 1A46

Fig. 4 Alignment of the deduced amino acid sequence of the B.

brevis (BB) and B. subtiIis (BS) degU genes. Identical and conserved amino acids are marked with asterisks and dots, respectively. The three conserved aspartate residues are boxed.

carrying the pML2 plasmid but were found to increase four-fold in the B. subtilis 1A311 strain, which carries the sacU(HY) mutation.

In-vitro expression of the degS-degU encoded polypeptides

The pML2 plasmid containing the degS-degU genes was incubated in-vitro with a coupled E. coIi transcription-translation system (Materials and methods). The labelled polypeptides were separated by SDS-PAGE (Fig. 5). Comparison of the polypeptides synthesized by the recombinant plasmid with those encoded by the vector plasmid pPLT03, and pPL708 (containing the cat gene with a functional promoter) indicated several ad- ditional bands, two of which had apparent molecular masses in agreement with those deduced for the ORFI- and ORF2- encoded proteins (respectively 45 000 and 30 000 Da). A discrepancy was noticed in the case of the ORF2 polypeptide between the experimental value and the one deduced from the D N A sequence. A similar disparity was previously recorded for the molecular mass of the degU gene (Kunst et al. 1988), and may r e -

BB BS

M N E Q V N E N K I Q I V I I ~ H Q L F R E G V K R I L A M E P E F E V V A D G E D G E N A V E L V E K Y N P D V I L M . . . T K V N I V I I I D D I H Q L F R E G V K R I L D F E P T F E W A E G D D G D E A A R I V E H Y H P D V V I

~

I N M P K V N G V E A T R D L I Q R Y P D V K V L V L S I H D D E S Y V T H V L K T G A S G Y L L K E M D A D A L I I N M P N V N G V E A T K Q L V E L Y P E S K V I I L S I H D D E N Y V T H A L K T G A R G Y L L K E M D A D T L I

E A V K V V A Q G G A Y I H P K V T H N L I K E Y R R L V N E D - E Q E S S E I G F K E V E Y R K P L H I L T R R E C E V L E A V K V V A E G G S Y L H P K V T H N L V N E F R R L A T S G V S A H P Q H E V Y P E I - - R R P L H I L T R R E C E V L

BB BS

Q L M T D G Y S N R M I G E A L Y I S E K T V K N H V S N I L Q K M N V N D R T Q A V V E S I K N G W V K V R Q M L A D G K S N R G I G E S L F I S E K T V K N H V S N I L Q K M N V N D R T Q A V V V A I K N G W V E M R

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Table 2 Effect of the degS-degU operon on the production of extracellular enzymes in B. subtilis (U units) Strains Enzyme specific activity (U/mg of protein)

Protease /3-Glucanase a-Amylase Levansucrase

(total)

Medium I a Medium IIb B. subtilis

1A46 (pML2) 1.20 18.3 0.85 0.78 2.68

1A46 (pPL708) 0.06 0.5 0.49 0.40 0.03

1A311 (pML2) 1.80 15.3 6.62 1.59 2.55

1A311 (pPL708) 0.17 2.6 1.60 0.30 1.95

"Cells grown in MMCH medium plus 2% sucrose (Kunst et al. 1988) b Cells grown in Luria broth plus 1% sucrose

Fig. 5 Identification of DegS-DegU polypeptides. Lanes: 1, vector pPL708; 2, vector pPL703; 3, recombinant plasmid pML2 containing both ORF1 and ORF2. Migration of standards and molecular mass in Kilodaltons is indicated in the left margin. Arrow- heads in the right margin indicate bands of approximately 45 and 30 kDa

flect the transcription-translation of the gene in a heter- ologous in-vitro system derived from E. coli rather than B. subtilis.

T h e 32.0-kDa protein that was considerably over- p r o d u c e d in p M L 2 but not in pPL703 or pPL708, was attributed to a fusion protein f o r m e d from read- through of an O R F d o w n s t r e a m of the degU gene into the cat gene.

Discussion

T h e degS-degU genes were isolated from an alkalophilic B. brevis and were selected for their ability to stimulate the p r o d u c t i o n of extracellular proteases.

W h e n on a high copy n u m b e r plasmid, the B. brevis degS-degU o p e r o n was found to be relatively stable and to cause the hypersecretion of several extracellular enzymes giving rise to possible industrial application. This p h e n o t y p e is similar to that obtained from B. subtilis strains carrying the sacU(HY) m u t a t i o n as well as to B.

subtilis strains overexpressing various accessory polypeptides, such as degQ or degR on multicopy plasmids ( A m o r y et al. 1987; Nagami and T a n a k a 1986; T a n a k a et al. 1987; Yang et al. 1986, 1987). Comparison of the increase in extracellular enzyme production in the presence of these genes has b e e n difficult to quantify, as in each case p a r a m e t e r s such as growth media, times of analysis and analytical methods have differed. H o w e v - er, from the published literature none has shown that a recombinant plasmid carrying the degS-degU genes increases extracellular enzyme p r o d u c t i o n in a B. subtilis sacU(HY) strain. On the contrary, Podvin and Stein- metz (1992) found that when a recombinant phage carrying the wild-type degU allele was introduced into a degU32(HY) strain, a partial suppression of the hyper- p r o d u c t i o n p h e n o t y p e associated with the degU32 mutation occurred.

T o our knowledge this is the first published sequence of a degS-degU o p e r o n to have been cloned from a Bacillus sp. o t h e r than B. subtilis. Sequence comparisons between the B. subtilis and B. brevis degS- degU regions show considerable homology t h r o u g h o u t the protein in a n u m b e r of conserved blocks in both genes. Within the DegS protein it was hypothesized that candidates for an autophosphorylation site were the Asp-152 and Asp-168 amino acids which may inter- act with the phosphate group of A T P through Mg 2+

salt bridges. This domain is located in the vicinity of the histidine residue (His-189), which is conserved in seven modulators (Msadek et al. 1990) and may be the site of autophosphorylation of DegS. Both the Asp-152 and the His-189 are present in the B. brevis degS gene.

T h e D e g U protein contains three aspartate residues, which are clustered to form an acidic binding pocket, as d e t e r m i n e d from the three-dimensional structure of o t h e r effectors. These are found at positions: Asp-10;

Asp-11 and Asp-56 and were found to be conserved in b o t h the B. subtilis and B. brevis D e g U proteins. In the B. subtilis D e g U protein the r e p l a c e m e n t of Asp-56 by Asn in the degU mutant abolishes the capacity of the D e g U protein to activate degradative enzyme synthesis (Msadek et al. 1990), and consequently, Asp-56 is pro- posed as the potential site of phosphorylation.

Primary structure modifications causing the mutational changes giving rise to the degS (HY) and degU-

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( H Y ) p h e n o t y p e s w e r e c o m p a r e d to t h a t o f the se- q u e n c e of t h e B. brevis d e g S a n d d e g U genes. N o c o r r e - s p o n d i n g c h a n g e s c o u l d be o b s e r v e d except in t h e region o f t h e degS200 m u t a t i o n w h e r e in B. subtilis G l y was r e p l a c e d b y Glu, w h i c h gave rise to the ( H Y ) p h e - n o t y p e . This p h e n o t y p e was f o u n d to b e d u e to t h e m u - t a t e d D e g S p r o t e i n being able to stabilize t h e D e g U - p h o s p h a t e ( D a h l et al. 1992; T a n a k a et al. 1991). I n t h e B. brevis D e g S p r o t e i n t h e G l y was r e p l a c e d b y A l a a n d the t w o a m i n o acids e i t h e r side of this m u t a t i o n w e r e also f o u n d to differ, W h e t h e r these a m i n o acid c h a n g e s could play a role in t h e ( H Y ) p h e n o t y p e o f the B. brevis d e g S - d e g U o p e r o n by stabilizing t h e D e g U - p h o s p h a t e in a similar m a n n e r w o u l d h a v e to be verif- ied b y f u r t h e r studies involving an in-vitro s y s t e m uti- lizing purified B. brevis D e g S a n d D e g U proteins.

Acknowledgements The authors wish to thank Ilze Meyer for her expert technical assistance and the Bacillus Genetic Stock Centre for providing the strains used in this study.

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