Gene, 49 (1986) 177-187

Elsevier

GEN 01840

The /I-glucanase gene from Bacillus amyloliquefaciens shows extensive homology Bacillus subtilis

(Recombinant DNA; nucleotide sequence; codon usage; start points; regulation of sucR -homology region)

Jiirgen Hofemeister =*, Andreas K&z”, Rainer RorrissP and Jonathan Knowlesb

177

with that of

transcription;

a Zentralinstitutftir Genehk und Kulturpjlanzenforschung der Akademie der Wtisenschaften der DDR, DDR-4325 Gatersleben (GDR) Tel. (+)37 4559250, and b VTT, Biotechnical Laboratory, SF-02150 Espoo (Finland) Tel. (+)358 0 4561

(Received September 16th. 1986)

(Accepted October IOth, 1986)

SUMMARY

The nucleotide sequence of a 1583-bp DNA fragment containing gene bgIA for endo-P-1,3-1,4glucanase (EC 3.2.73) of Bacillus amyloliquefaciens strain BE20/78, a high producer of secreted enzymes, has been deter- mined. The gene bglA comprises an open reading frame (ORF) of 717 bp ( = 239 codons) starting with ATG at 469 up to the translation stop codon TAA at 1188. Upstream from the translation initiation codon ATG, the ribosome-binding sequence 5’-AAAAAAGGGGG-3’ and two putative bgL4 promoters have been identi- lied. A box of eleven AT out of twelve base pairs (bp) precedes the -35 region of promoter Pl. Beyond the translation stop codon UAA, a sequence of 69 bp can be folded into a hook-like stem-loop structure which probably functions as a transcriptional terminator. The ORF region of the gene bglA reveals about 90% homology with another fl-glucanase gene, bglS of Bacillus subtilis Cl20 sequenced by Murphy et al. (1984). Three regions of frequent amino acid (aa) changes are indicated. However, the major difference between these is a set of deletions within the non-coding region separating the bgL4 gene from an unknown preceding ORF and by one deletion shortening the proposed signal peptide by three aa (Pro-Tyr-Leu-). The putative transcription terminator of gene bglA completely lacks homology with a B. subtilis bgIS gene. The signification of deletions erasing the ‘sacR -homology region’ in B. amyloliquefaciens, which have been detected in proximity of the /I-glucanase gene of B. subtilis by Steinmetz and Aymerich (1986), is discussed.

* To whom correspondence should be addressed.

Abbreviations: aa, amino acid(s); bp, base pair(s); bgiA, gene coding for E. amyloliquefaciens p-glucanase; bglS, gene coding for

B. subtih b-glucanase; dd, dideoxy; del, deletion; IPTG, iso-

propyl-B-D-thio-galactopyranoside; kb, kilobase or 1000 bp;

nt, nucleotide(s); ORF, open reading frame; PolIk, Klenow

(huge) fragment of E. coli DNA polymerase I; RBS, ribosome-

binding site; TY medium, see section b of MATERIALS AND

METHODS; u, unit(s); XGaI, 5-bromo-4-chloro-3-indolylji-

galactoside.

INTRODUCTION

The type and amount of a-amylase produced by industrial strains of B. amyloliquefaciens has been claimed to be the only consistent phenotypic character that separates this taxon from B. sub&s (Priest, 1981). In confirmation, no apparent homo- logy has been established at the nt level, by com- paring the cloned genes (Yang et al., 1983; Yamazaki et al., 1983; Takkinen et al., 1983).

0378-l 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

178

The fl-glucanases (endo-/?-1,3_l,Cglucanases) of B. subtilis and B. amyloliquefaciens are both charac- terized by a substrate range similar to lichenase (EC 3.2.1.73) of germinating barley (Anderson and Stone, 1975; Boniss et al., 1980). However, the P-glucanase activities secreted by various strains of both taxa showed considerable variability (Borriss and Zemek, 1980; H&We, 1984).

Recently, we reported the cloning of a /3-glucanase gene of B. amyloliquefaciens strain BE20/78, a high producer of the enzyme, which is used for industrial applications (Borriss et al., 1985). The restriction map of the bglA gene pointed toward identity with a &glucanase gene cloned from B. subtile strain NCIB8565 (Hinchliffe, 1984) but indicated variabili- ty at the nt level when compared with a /?-glucanase gene cloned from B. subtilis Cl20 (Cantwell and McConnell, 1983).

In this paper we report on the nt sequence of gene bgk4 of B. amyloliquefaciens strain BE20/‘78, which

codes for secreted #&glucanase, and on sequence comparisons with a recently sequenced bglS gene of B. subtilis strain Cl20 (Murphy et al., 1984). Extended homolo~ within the translated region of the genes contrasts with striking differences in the regulatory regions preceding and terminating the bgl genes.

adjusted to pH 7.2). For growing competent cultures, SOB and SOC media were used according to the recipes given by Ham&an (1983). After transfor- mation, cells were plated into H-top agar onto M9 salt medium supplemented with IPTG and XGal, according to the recipes given in the Ml3 Cloning and Sequencing Handbook (Amersham Inter- national, 1984). Recombin~t phages carrying the fi-glucanase gene were identified by plating on lichenin agar (i.e., M9 salt medium supplemented with 0.2% lichenin isolated from Cetraria islandica as described by Borriss et al., 1980). Glucanolysis of lichenin was then visualized after plaque formation by flooding the plates with 0.1% (w/v) solution of Congo Red for 15 min. Light haloes are indication of /?-glucanase production by recombinant phages.

(c) Enzymes

Restriction endonucleases, T4-DNA ligase, Polfk, exonuclease III of E. coli and T4-polynucleotide kinase were purchased from Boehringer Mannheim; Sl nuclease and T4-RNA ligase from Bethesda Research Laboratories. In some ligation experi- ments, LIGAITTM from P&S Biochemicals Limited, Liverpool, U.K., was used. The enzymes were applied according to the manual of Maniatis et al. (1982).

(d) T~sf~ti~ MATERIALS AND METHODS

(a) Bacterial strains, phages and plasmids

Plasmid pEG1 (8.0 kb) cont~~g, in a 3.6kb DNA fragment, the gene bgL4 coding for secreted /?-glucanase of B. amyloliquefaciens strain BE20/78, a derivative of strain ATCC15841 (Borriss et al., 1985), was used for subcloning the fi-glucanase gene into the Ml3 phage vector mpl0 (Messing, 1983). The Escherichia coli host for transfection of plasmid and template DNA isolations was JM 10 1, supE, thi, d(laq-proAB), [F’fraD36, praAB +, lacPZAMl5) (Messing, 1983).

(b) Media, plating and assay of /I-glucanase activity

Transfection of competent E. coli cells was per- formed according to the procedure of Hanahan (1983).

(e) Plasmid and template DNA isoiations

Ml3 RF DNA was preparedfrominfected JMlOl cultures by the cleared lysate method (Clewell and Helinski, 1970; Clewell, 1973). Rapid isolation of phage RF DNA as well as direct gel electrophoresis of Ml3-infected cultures was made as described by Messing (1983). Single-stranded template prepara- tions of Ml3 phages were done as described in the M 13 Cloning and Sequencing Handbook (Amer- sham).

Bacteria were grown in 2 x TY (16 g Bacto- tryptone, 10 g yeast extract, 5 g NaCl per liter

(f) Deletion method

179

RESULTS AND DISCUSSION

For targeting deletion breakpoints within cloned Bacillus DNA, the protocol of He&off (1984) was followed, with a number of modifications: (1) PstI was used to create 3’-protrudiig ends for protection of the primer site and Hind11 cutting for targeting exonuclease III digestion; (2) the PolIk reaction was followed by treatment of DNA samples in 20-~1 volumes each with 1 u of TCpolynucleotide kinase, according to Maniatis et al. (1982) for 15 min at 37°C because of low ligation efficiencies; (3) the kinase reactions were supplemented after 10 min of 65°C treatment with T4 DNA ligase and T4 RNA ligase (100 u and 1 u, respectively) and incubated for 18 h at 21’ C; (4) before transfection, samples were deproteinated by phenol and chloroform extractions and used after extraction with ether.

(g) Nucleotide sequencing

The dideoxy chain termination sequencing proce-

dure of Sanger et al. (1977) was employed essentially as described in the Ml3 Cloning and Sequencing Handbook (Amersham). Individual plaques were picked and purified once by plating appropriate dilutions with JM 101 before phage clones were used for single-stranded template DNA preparations.

The 17-mer sequencing primer was from UMIST, Manchester, U.K. The ddNTPs were purchased from Boehringer Marmheim, and individual working solutions were established: ddATP, 37.5 PM; ddCTP, 40 PM; ddGTP, 100 PM; ddTTP, 500 PM.

The label used was [ a-35S]dATPaS (SJ.304)from Amersham. Usually, eight or ten reactions were done in parallel in microtiter plates at room temperature and placed, after addition of formamide dye mix, into a sand bath for denaturation for at least 5 min at 90 or 95°C.

Electrophoresis was carried out using 6% polya- crylamide gels (40 x 20 x 0.02 cm) essentially as described in the Ml3 Cloning and Sequencing Handbook (Amersham).

(a) Subcloning, generation of deletions and sequenc- ing

A 2.4-kb DNA fragment containing the entire /?- glucanase gene of B. amyloliquefaciens BE20178 was subcloned from plasmid pEG1 (Borriss et al., 1985) by cutting with EcoRI + BglII and ligation into Ml3 vector phage mpl0 (Messing, 1983). Phages carrying the bglA gene were screened after transfection by plating on lichenin agar and staining with Congo Red in order to visualize glucanolysis due to P-glucanase production of infected E. coli cells. The DNA of a bglA + -phage M 13 mplO-42 was isolated and, after confirmation of the restriction map shown in Fig. 1, DNA was used for generation of targeted deletions according to the protocol of He&off (1984). About 75% of the phages screened according to the exo- nuclease III procedure and transfection were found to contain deletions suitable for sequencing reactions (see section g of MATERIALS AND METHODS).

Finally, a spectrum of about 30 phages was obtained that showed overlapping deletions covering the gene sequence within the 1583-bp Bacillus DNA fragment (Figs. 1 and 2). From that deletion, of the clones listed in Fig. 1, only phage 5312 carrying about 213 of the bglA gene coding region showed preserved fi-glucanase activity.

(b) The bglkcoding sequence

The sequence presented in Fig. 2 contains only one ORF sufficient to code for a protein, which would be about the size of the secreted /I-glucanase of B. amyloliquefaciens (Borriss et al., 1985). The reading frame, starting from bp 469 with ATG and extending up to bp 1185, spans the EcoRV site, which has previously been shown to lie within the j?-glucanase gene coding region; the reading frame also corresponds to the bglA gene region localized by Tn5 transposition mutagenesis (Borriss et al., 1985).

The deduced protein coded for by the 717-bp region, comprises 239 aa residues, corresponding to an A4, of 26 950. The calculated molecular weight of the putative pre-/3-glucanase is thus about 3 kDa larger than that calculated for the exoenzyme. It is therefore suggested that a signal peptide of about 26 aa residues is processed during enzyme secretion, as has been shown for many other secreted proteins.

180

M13mplO-42 Eh HI 1 I ,

Cl

Pllage clones 4

3R, QK.15 612 ,lOB

2&f%? ml6.6/1,&$

74.32.38 5.7

2M ,d

I

A

E5 PI HII HI A2 H2 Et+i1 m.cs. PS

rl 4 . r

A2 Sl

WAgene t 1320 bp

G

Fig. 1. Physical map of the 2.4-kb DNA fragment of E. amyloliquefuciens cloned into M13mplO vector. The upper panel shows

overlapping deletion templates generated and chosen for sequencing. The proposed position of the translation start codon ATG (see Fig. 2), the multiple cloning site (m.c.s.) and primer annealing site (PS) of the phage are indicated. Restriction sites are: Cl, ClaI; Hl, HinfI; A2, AvaII; E5, EcoRV; Sl, SphI; P, PvuI; H2, HincII/HindII; B2, EglII; BHl, BarnHI. The &glucanase activity of the clone in its E. coli host is also indicated by ( + / - ) marks in the figure.

(c) Specificity of the bgl genes

The B. amyloliquefaciens bglA gene shows striking homology with a &&xxnase gene of B. subtilis (which we will name by convention) bglS recently cloned from B. subtilis strain Cl20 by Cantwell and McConnell (1983) and sequenced by Murphy et al. (1984).

(1) Codon usage and amino-acid composition As shown in Table I, there are minor differences

in codon usage between bglA gene from B. amyloli- quefaciens and bglS from B. subtilis. The invert fre- quency of Pro codons CCA and CCG, as well as other.speciticities in codon usage in the B. amyloli- quefaciens and B. subtilti bgl genes, is indicated (!) in Table I. The aa Gly and Thr are most abundant, the frequency of Lys and Pro is equal, while the number of Leu and Ser residues varies most in the fi-glucanase precursor from B. amyloliquefaciens and B. subtilis.

(2) Homology between pre-fl-glucanase Comparing the putative signal peptides of both

A 9-bp deletion (del9a, Fig. 2) at the translation Bacillus strains (Fig. 2), seven aa changes are, due to

start shortens the deduced B. amyloliquefaciens pre- a number of substitutions, clustered within the hy-

/I-glucanase protein, in comparison with the B. sub- tilis precursor protein by three aa residues (Pro-2Tyr- 3Leu-4), thereby lowering the hydrophily of the very N-terminus of the putative signal peptide. Provided that the same processing site of the signal peptide is used in B. subtih and B. amyloliquefaciens, and taking into consideration the calculations made by Murphy et al. (1984), the signal peptide of bgL4 coded /I-glucanase should end with Val-23 or Ala-25. Indeed, by the dansyl chloride method of Gros and Laboness (1969), the N-terminal aa of highly purified B. amyloliquefaciens /I-glucanase was found to be either Glu or Gin (R. Jung and G. Jttttner, pers. communication). The putative signal peptide clea- vage site of B. amyloliquefaciens /_I-glucanase is there- fore most probably at the bond between Ala-25 and Gm-26 (Fig. 2). The last eight residues of the signal peptide are small aa (Gly-18-Ile-Thr-Ser-Ser-Val- Ser-Ala-25), and the Ala-25-Gin-26 cleavage site is compatible with the empirical rules (e.g., -3,-l rule; window of cleavage site) constructed by Von Heijne (1983, 1984) and Perlman and Halvorson (1983).

181

.,EcoRI

E F N*E E S’L Ii V v* R F VsT H L*K F F ;7” Q R L*F N G*T ii M ; S Q D’ ~~ACGAAG~(3TCGCTTCRCT~TT~TCGGTT~GTC~CCC~~TTR~~GTTTTTCG~G~~G~GT~T~TTT~~CGGC~CT~~~~lGG~~~GCC~~G~CG --~~-~---~---~~----- G_______-___p__-__--____-____----_-_--~~________~_____---_-_~--_-_-----___-~____

SUB AM\’ .,_ = D E = z = = = = = = = = = I = =? =n__;===;====.=

* * * iI l t * * * x *

D F L L D T U K E I( V H R F, V E C T I( C-.1 Q T-\’ I E i E V E.-Ii t L- SUB FITTTTTTGCTGG~TRCRGTG~~~GA~~~GT~T~~T~G~G~GT~TG~RTGC~~G~~G~~~~TC~R~RC~T~~~TTG~GCGGG~GT~TG~G~~C~~GCTC~C &MY ----__-~---_-G---_____-____________-k6_---___-G--_T__---___

= = = = E=======Q==K===N=H===_K=IG=E=

1:: 101

34

SUB CIMV

SUB WlV

268: 201

67

T S D E L L \‘- L T I .H I E .i U U C- Q A - - CI~GTG~CGAGCTGCTGTRTTTAACCkTTC~C~T~G~R~GGGT~GTT~~~~~~GC~T~~TG~G~G~G~TG~C~TTTGTGTTT~~TTGTGTT~~CTTTTTCT ~_--__-----___-------___6__----_-------_______~___-__-~__-----~_----_d,l 11~ ____--C-_*__*-T-G____-

5 = = = = = E = = = = = = = = = 2 = II =

* * * * I: * * *

* Region of honology,with sac,” - - - - - - - - _ _ _ _ _ _ _ _ a

* * * I _ _ _ _ _ _ _ _ _

TACc)TTCACkTRTG**A*TGGT*GG~TTGTTACTG*T*~~G~*GG~~**~~~T**~TTG~~*TG*GTG~,jG~T~~T~T~TGT~TGTG~T~*TGGT~*TT~ _C__-___T___ ____-_---__-G-_-______________--- l

f l-de1 9-l * ,

i * d_l ?:: ,-_---,--_-------__-G---

x

301 230

SUB hMV

SUB AM\’

SUB clMV

401 358

50: 416

1

~GGTTTTT~;TTTTTTC~G~GGG~*G~TG~TG~T~G?T~~*GG*TT~*~~TT~GT*~G*~T~,j*T~TT~~~*TT*TTTT~*~~G*TGTT~~~TTTTG**~ _-_---G____---R+-_-fi-_

de1 42 * * f

_----___-----~-;-_------_~~ *

Pi-35 P2-35

* I l I: 6 < M P* V L K ‘H 0 L I! L L II* T 6 L

GACITCRTGTAAGClTCCllC~T~G~~~~CGCTTTCTGG~TTGTT -G-___----T____--T-*__---G--TG_----~*__G-----__--T-_~ fi-----__-_----__-fi*----_-----C___------

rdel9a+ M = = = = = , = = = = = * * * II * * I: *

Pi-10 R.B.S.

*I F M S L F A VP T A T fi S R z T Ii G* 5 F F *D P F i G V 11% S G F *W 0 K A”

* I TRTGAGTTTGTTTGC~GTCACTGCT~CTG~CTC~G~TC~~~~~GGTGG~TCGTTTTT~G~CCl:TTTT~~~GGCT~T~~~TCCGGTTTTTGGC~R~A~GC~ __--------- G--GG~_----T___G_-TT__6_-_--_____--~____----__-T__~_----____~---____------G_-~---___-----T

6:; 587

13

D G Y* S N G *El M F ;: C T W* R A N *N U *

S M T S L*G E M*R L A L” T S P* SUB GATGGTT~TTCG~ATGG~CIFITF1TGTTCAACTGC~CGTGG~GGGCT~~T~~CGT~T~~~TG~~GT~~TTGGGTG~~~TGCGTTT~GCGCT~~C~~GCC~~G CIMV -_------ ~__~___--_G-__----T___-----T_-___T_-___----_-~--T_---_----_-~-__-----____--G--___G_---_T__GT

E a 1 E E = n========================== li x * 1: ): * * * * *

.~_.~ _______~ _ _

7;; 687

47

888: 787

8%

SUB WIV --_-___------_---_----~_---_--- ~--r,_--_-------___--_--__-_----____--_-_____-_-_~___---_____---___~__

s======================5==.====1=

* I * * * 1: * * * l

I 1 EEOR”

U S S : F T V’T R P*T D R T” P I.,1 D*Q I D*I E F L* G K H*T T K*U Q F ;II SUB TTCATCGTTCTTC~CTTRCFCACGTCC~~C~G~TCG~~CTCCTTGGG~TC~G~TT~G~~TTTTT~GG~~~~C~~~C~~CG~~GGTTC~~TTT~~C AMV ----__-_--------_-T---G-_-----G_-GG-G-___---_-__-G--_-___----____----G______G______---___-----____--

1 = D I = = = =G==EG====E========D======= I 1 *L * % f * * * *

116 981 807 113

150 1001 307 147

i x )I I I lAvaI s x * * * *I V V T N G fi G N H E k I U b L 6 F D R fi N A V H T V I? F D C Q P N

%t T~TTFITkCRIIFtTGGTGC~GGPIRFICC~TG~GA~G~TTGTTG~T~T~GGGTTTG~TGC~G~~~~TGl~~T~TCRT~~GT~TG~~TT~G~TTGC~~GCC~~~CT ----__----_--_~------_---_---____T-c_cG-_-~------__---_--__-_---__-------__G_----__-G_-____----

= = a = E a I I = I =

l * *

,I f -a f *= = = ; = = =* E = = *= = = ; = i =* = =

* * * l i l II .* * *

S I I( W V U D G Q L K H T A i El Q I P T T L i K I M M N L C N G T SUB CTPTCAARTGGTPTGTCG~CGGGC~~TT~~~~C~T~CTGC~~~~~~~~~~~TTCCG~~~~~~t:TTGG~~~G~Tt:~TG~TG~~~TTljTGCR~TGGC~CGGG NIV _---T-______-______T_-___---__--_----_-----_-~----__~___,j__,j_,j_~,~__G__~___________T_____G_____T_____

r 5 = = = zz = E E = = = = = = T===(qfiF==z====W=== * 1 * * L * .!I. * x

183 ii%1 1807

180

SUB RMV

216 G ” D E W L G 3 V N 6 U N P L V k H V D id U R V R K K - 1201 TGTCGRTGA~TGGCTTGGCT~CT~C~~TGGTGT~~~T~CGCT~TR~GCTC~TT~Tlj~~TGGGTGCGCT~T~G~~RA~R~T~~TGCC~RATGTGR~~G~~C 1187 ___T--___T----_~__T-_-_____---C_____---__~____-_____---,~____--~_____---____-__-___-__T~~_G~~~~GG_TTT- 213 I = I D====nlrn=EI=rE===“==los-

1301 1207

\ ~-*\*-l, t * * * * SUB CTGCTGC~~TAT~GCRGCCTCTTATG~TTGT~~TG~C~~GTT~TTGlj~~TG~~T~TTCT~TTC~CTC~T~~T~TC~T~TTTG~TCTTCTCCCTCTGT~~~ AMV GCTGGCGG-RTCCTTTTTTGCI---PFI-CGRA-T~~T-C~R~-C~~~~GCGC~G~~~--GT~~TT~~~--CG-TljT-C~C~~-G~-~~T-TG~-GGTG--T

1*- .I * * * .* * I T -

SUB TCACGTAC WV -7TT

*

1401 1387

Fig. 2. The nucleotide sequence of the B. amyloliquefuciens B-glucanase bgL4 gene region (AMY) is compared to a previously published

sequence of a B. subtih DNA fragment (SUB) encoding the /I?-glucanase bglS gene (Murphy et al., 1984). Both sequences begin from

a homologous EcoRI site at the 5’-terminus. The aa sequence deduced is shown in single-letter code for a preceding unknown reading

frame ‘x’ and the open reading frame of the &glucanase gene. The nt and aa changes are indicated. A set of deletions in the order dell 1,

de19, de123, de142 and del9a characterizes the DNA upstream from the B. amyloliquefaciens bgL4 gene region. The putative promoters

PI and P2, and RBS are underlined. The region of homology with sacR, including the inverted repeat region in B. subtih DNA, is shown

within the B. subtilis DNA (---). Regions of dyad symmetry are indicated by arrows.

182

TABLE I

Codon usage in the fi-glucanase genes a

Codon Amino Number Codon Amino Number

acid of acid of

residues residues

Codon Amino Number Codon Amino Number

acid of residues acid of residues

Ba Bs -

Ba Bs Ba Bs Ba

uuu Phe 9 9 ucu Ser 4 2 UAU Tyr 12 14 UGU cys 1

uuc Phe 4 5 ucc Ser 2 2 UAC T yr 4 3 UGC cys 2

UUA Leu 3 3 UCA Ser 3 4 UAA End 1 1 UGA End 0

UUG Leu 6 5 UCG Ser 4 3 UAG End 0 0 UGG Trp 8

cuu Leu 2 5 ecu Pro 2 3 CAU His 4 4 CGU Arg 2

cut Leu 2 1 ccc Pro 0 0 CAC His 0 1 CGC Arg 2

CUA Leu 1 2 CCA Pro 2 !4 CAA Gln 6 6 CGA Arg 1

CUG Leu 1 2 CCG Pro 5! 2 CAG Gln 1 2 CGG Arg 0

AUU Ile 4 3 ACU Thr 5 6 AAU Asn 8 8 AGU Ser 3!

AUA Ile 2 !O ACC Thr 2 !O AAC Asn 10 12 AGC Ser 1

AUC Ile 3 4 ACA Thr 8 11 AAA Lys 10 9 AGA Arg 2

AUG Met 9 8 ACG Thr 5 5 AAG LYS 3 4 AGG Arg 0

GUU Val 5 4 GCU Ala 5 6 GAU Asp 10 8 GGU Gly 6

GUC Val 4 5 GCC Ala 2 3 GAC Asp 3 4 GGC Gly 4

GUA Val 1 2 GCA Ala 4 6 GAA Glu 5 5 GGA Gly 7

GUG Val 1 2 GCG Ala 4! 1 GAG Glu 3! 1 GGG Gly 7

Bs

0

!4

0

6

3

1

2

1

1

1

2

0

7

4

7

4

a The data for B. sub&s are evaluated from published sequence data (Murphy et al., 1984). The AUG initiation codon is included with

the Met. End = terminator codons.

+3

I A B +2 . . -_

4+1- .- ,. Y ,u .-

E

O-r 3

3 . I

-l- -

. . . . . ._. . m . ._.. .

I 100 2oo C-terminus

I 1 t

Amino acid residues

Fig. 3. The hydrophobicity profile ofB. amylofiquefaciens pre-Sglucanase was calculated according to Hopp and Woods (1981). Arrows

and dots, respectively, indicate regions and aa residues where variability is detected in comparison with /%glucanase of B. subcilia of which

the hydropathy profile is not shown.

183

drophobic portion of the signal peptide next to the cleavage site. Including the aa residues deleted from the hydrophobic portion, a total of 10 out of 28 aa within the putative signal peptide region of B. subtilis 8-glucanase are altered with respect to B. amylolique- faciens /?-glucanase.

Within the region of gene bgL4 coding for the ma- ture /?-glucanase, 62 additional bp substitutions re- sulted in 21 aa changes within the mature enzyme, and thus yield homology of about 90% both at the DNA and the protein level. These aa changes also tend to be clustered (see Fig. 2).

The hydrophobicity profile of B. amyloliquefaciens is shown in Fig. 3.

In general, the B. amyloliquefaciens /?-glucanase protein sequence, when compared to that of B. subtilis, shows a number of more positively charged regions. Region C is particularly distinct in this sense. Amino-acid substitutions occur mainly in regions of roughly neutral charge, and only very rarely in highly hydrophilic regions.

The variation between the pre-j?-glucanases of the two Bacillus strains thus appears to be most pronounced within the putative signal sequence region, while homology in the mature /.?-glucanase coding sequence is somewhat higher than homology of secreted neutral and alkaline proteases of B. subtilis and B. amyloliquefaciens, which are availa- ble for comparison and are estimated to give 82 and 86% homology, respectively (Vasantha et al., 1984; Yang et al., 1984; Stahl and Ferrari, 1984; Wong et al., 1984). The pre-pro-sequences of the proteases, however, are also distinguished by lower level of homology (of about 76 and 75 %, respectively), than the mature exoenzyme indicating the trend for a less conserved maintenance of pre-pro-portions of secreted enzymes in Bacillus (Sibakov and Palva, 1984).

(d) The transcription unit

Murphy et al. (1984) have scanned the sequence preceding the bglS gene coding region and identified ‘consensus’ regions of Bacillus a55 promoters (Moran et al., 1982; Murray and Rabinowitz, 1982; Johnson et al., 1983). In Fig. 2, it can be seen how these putative promoters PI and P2 are arranged within the B. amyloliquefaciens bgL4 transcription unit. The promoters PI and P2 overlap in a region

77 bp upstream from the translation start codon ATG. A more distant promoter region, proposed in B. subtilis to span positions 435-480 (Murphy et al., 1984), has been erased in B. amyIoliquefaciens by the de242 deletion (Fig. 2).

An AT-rich region of about 30 bp upstream from the B. subtilis promoter PI-35 region is reduced in length in B. amyloliquefaciens due to the de142 dele- tion (Fig. 2). AT-rich regions have been shown to affect the activity of many promoters (Moran et al., 1982; Doi, 1984); it is likely that this variation will affect bglA gene expression in B. amyloliquefaciens.

Substitutions of two and three bp are found within the consensus regions of the putative promoters Pl and P2, respectively. The transcription initiation site for RNA polmerase within the bgL4 gene sequence has not yet been determined. According to the rule that the mRNA start points are predominantly purines, and some 6-9 bp beyond the designated T nt in the -10 region (Rosenberg and Court, 1979), bgL4 gene transcription most probably starts from one of the A residues 6 or 9 bp downstream from the Pl-10 region, which would give a 34 or 37-nt leader

sequence. The sequence downstream from the bglA gene

ORF contains a set of incomplete inverted repeats which can be folded into an &shaped structure, of which two possible configurations are demonstrated in Fig. 4. Both forms are distinguished by stem II and by their calculated free energy AG of -23.7 and -21.3 kcal/mol, respectively. The stem I resembles Rho-independent transcription terminators of E. coli (Platt and Bear, 1983) in which a short G + C-rich segment in the centre of dyad symmetry is followed by a string of U residues in which transcription is usually terminated. The string of U residues lies within the turn of the S-like stem-loop structure, and pairs partly with a 3’-terminal CAAUCAAA se- quence which has been proposed to be a common sequence of Rho-dependent terminators in E. coli (Rosenberg and Court, 1979; Platt and Bear, 1983).

The region of the supposed transcription termina- tor in B. amyloliquefaciens completely lacks sequence homology with the T-shaped structures found by Murphy et al. (1984) downstream from the B. subtilis bglS gene sequence, which they interpreted to have a role in attenuation-like modulation of transcription of a gene downstream from the bglS gene. Thus, there is pronounced variation in those transcription

184

u 625)

: -c" C G- E

C-G C-G

u" G

- A stem1 E G

-A sbm1 U-A U-A A-U A-U G-C G-C G-C G-

(1) A-U

YI

(11 A- AAAUIJUGUACAGAAA - U

(13) - AG -3’ ~U~~GUACAG~3 j

U-A U-A U - A stem11

dG= -23,7 kcalhol G - C A -u u _-A U-A

c :: (69) u - AG -3’ u- u- U-

A= -21,3 koal/mol G -

u^- U-

(45) A Cl (45) A - U

A” C

C :: t A U AC GA

C G AA

AAAU

Fig. 4. Predicted secondary structures ofthe 3’-terminus ofbgL4 -mRNA due to the inverted-repeat sequences. The calculated free energy AG of the configurations is indicated. The translation stop codon, the U-bulge region and a CAAUCAAA sequence are underlined.

terminator-like structures in B. subtilis and B. amylo- liquefaciens bgl gene regions.

(e) Translation initiation sites

A putative RBS on the bglA-mRNA, comple- mentary to the 3’-end of 16s rRNA of B. subtilis 3’-UCUUUCCUCCACUAG-5 (McLaughlin et al., 1981), was detected 9 nt upstream from the translation start codon AUG within the sequence 5’-AAAAAGGGGGaAUGcUAAcAUG-3’ (low- er-case letters are to stress the codons AUG and UAA within the ‘window’, see below). The trans- lation initiation region differs from the corresponding region of B. subtilis bglS-mRNA by five changes. The transversion G to A at position -14 (counted from the first A of the initiation codon) might shift the centre of the pairing region (UUUCCUCC). The two other transitions might change the relative

stability of the Shine-Dalgarno interactions charac- terized by the free energies AG of -18.8 kcal/mol in bgZS-mRNA of B. subtilis, and of -16.6 kcal/mol in bglA-mRNA of B. amyloliquefaciens. Within the spacing between the last nt of the RBS and the first nt of the AUG start codon, two nt changes are found, of which one constitutes, in bgL4 -mRNA, an

UAA stop codon next to the start codon. Increased complementarity of the RBS (Gold et al., 1981; Band and Henner, 1984), and the frequency of A’s and T’s preceding the translation initiation codon, might contribute to increase translational expression of the bglA gene in B. amyloliquefaciens, as compared to B. subtilis.

(f) The ‘sacR homology region’

Murphy et al. (1984) detected a 6 l-m-long invert- ed repeat 92 bp beyond an unknown reading frame ‘X’ and 67 bp upstream from the Pl-35 promoter region of gene bglS (Fig. 2). Within the inverted repeat, 2 x 26 nt could pair to form a stem-loop structure with a AG of -18.9 kcal/mol. The inverted repeat has now been identified to build the centre of a 98-nt sequence homologous to sacR of B. subtilis (Steinmetz and Aymerich, 1986). The sacR region maps close to sacB and has been found to regulate the inducibility by sucrose of sacB, the structural gene of levansucrase (Steinmetz et al., 1985). These authors argue that sacR functions as a transcription terminator via attenuation of sacB gene transcription in response to a diffusible regulator whose activities are controlled by sucrose.

185

On the B. subtilis chromosome, gene bglS has been localized between SUCA and purA close to the marker

sacs (O’Kane et al., 1985; Borriss et al., 1986). The neighbourhood of bglS and sacs lead Steinmetz and Aymerich (1986) to suppose that sacs might be identical to the ‘sacR-homology region’ close to the bglS gene and that this region codes for a small RNA able to hybridize with the transcript of sacR, the transcription attenuator of sacB. According to these speculations the ‘sacR-homology region’ located close to the /?-glucanase gene bglS in B. subtilis might therefore not necessarily be related to bglS gene regulation. This is, however, indicated by the fact that, in B. amyloliquefaciens, four distinct deletions, dell 1, de19, de123, and de142, are positioned in just that ‘sacR -homology region’ upstream from the bglA gene promoter (Fig. 5). We are, therefore, inclined to assume that these deletions are not random, but are related to the ‘sucrose response’ and/or peculiarities of bglA gene regulation in B. amyloliquefaciens. To our knowledge, the metabolism of levansucrase has not so far been extensively studied in B. amylolique- faciens. Production of /I-glucanase has however been shown to be 20-50-fold greater in the B. amylolique-

faciens strain (a derivative of ATCC15841) used for bglA gene cloning (Borriss et al., 1986; Borriss and Hofemeister, 1985). Moreover, a bgl gene cloned from B. subtilis strain NCIB8565, which was chosen

Fig. 5. Topology ofsacR location within the B. subtilh sacB gene region with its counterpart, detected by Steinmetz and Aymerich (1986), in proximity to the bgZS gene, and reconstruction of its rudiment in B. amyloliquefaciens bgL4 gene region, which is affected by a set of deletions (dell 1, de19, del23, de142 and del9a). The site of a putative RBS at the 5’-end of the ‘sacR stem-loop structure’ and the gene promoters upstream of the respective sacB, b&S, or bgL4 gene ORFs are shown. The nt are numbered according to published sequencing data (Steinmetz and Aymerich, 1986; Murphy et al., 1984).

TABLE II

Amino acid composition of /I-glucanase precursor proteins of

B. amyloliquefaciens (Ba) and B. subtilh (Bs) The data for B. subtih are from Murphy et al. (1984).

Amino acid

Number of residues

Ba Bs

Amino acid

Number of residues

Ba Bs

GUY Thr Asn

Ser

Tyr Leu Ala Phe

Lys Asp

24 22 Val 20 22 Met 18 20 Pro 17 13 Ile 16 17 Glu

15 18 Trp 15 16 Arg 13 14 Gln 13 13 His

13 12 Cys

4

3

13 8 9

by Hinchliffe (1984) because of its high B-glucanase production in comparison with other B. subtilis strains, seems according to its restriction map to be closely related to, if not identical to, the B. amyloliquefaciens gene bgL4 described in our studies. We therefore suggest the high level /I-glucanase production to be linked with the source of the gene. Thus, we expected, after retransfor- mation of recombinant plasmids containing either gene bglS of B. subtilis 168 or bgl4 of B. amylolique- faciens BE20178 into B. subtilis, to get either the ‘high’ or ‘low’ producer status encoded by the respec- tive recombinant plasmid. However, we found that

B. subtilis transformed with the bgL4 gene either on a plasmid or (via an integratable plasmid) inserted into the chromosome, expressed only about twice the /_I-glucanase level of transformants containing the bglS gene insert (R.B. and J.H., unpublished). From these observations we are inclined to speculate that the ‘super’ potency of B. amyloliquefaciens to secrete B-glucanase might be due to a positively acting regu- latory element which is absent from B. subtilis, and whose regulatory function might require the presence of the sequences replacing the ‘sacR-homology region’ in front of the B. amyloliquefaciens bglA gene. Alternatively, one might assume an enhancer effect of deletions due to the elimination of a potent tran- scription terminator, thus allowing readthrough into the bgL4 gene region in B. amyloliquefaciens.

186

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This work has been partly realized within the frame of cooperation between the Academies of Sciences of Finland and of the German Democratic Republic. J.H. would like to thank professor Tor- Magnus Emu-i, especially colleagues of the Recombi- nant DNA Group for support and hospitality. We wish to acknowledge contribution made by Drs. Seitz and H. Schutz (for Fig. 4 and section e, respec- tively), by G. Jtittner and Dr. R. Jung (for section c), and by Dr. H.-W. Jank (for computer analysis of sequencing data for Fig. 2). We are also indebted to Dr. Steinmetz for providing us with data discussed in section f prior to its publication.

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Communicated by Z. HradeEna.