Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens

Gene. 96 (1990) 89-93 Elsevier

89

GENE 03824

Isolation and characterization of ievansucrase-encoding gene from B a c i l l u s amyloliquefaciens (Phagemid; recombinant DNA; sucrose regulation; signal sequence)

Leslie B. Tang, Reijer Lenstra, Torben V. Borehert and Vasantha Nagarajan Central Research and Development Division. E.L duPont de Nemours Company, Wilmington, DE 19880.0228 (U,S.A.) Received by R.E. Yasbin: 29 May 1990 Revised: 14 August 1990 Accepted: 17 August 1990

SUMMARY

The gene encoding levansucrase (LVS) from Bacillus amytoliquefaciens (sc~gB[BarnP]) wa~ isolated, sequenced and expressed in Bacillus subti~lis. Analysis of the nucleotide sequence of sacB[BamP] reveals extensive homology with that of the B. subtilis LVS-encodiag gene in the promoter and coding region. The sacB[BamP] gene cloned in a multicopy plasmid is induced by sucrose in B. subtilis.

INTRODUCTION

Sucrose metabolism in B. subtilis has been extensively studied by several laboratories (Lepesant etal., 1976; Steinmetz ¢tal., 1985; Shimotsu and Henner, 1986). Levansucrase (sucrose: 2,6-/kD-fructan-6-/~-D-fructosyltransferase.: EC 2.4.1.10) is secreted by B. subtilis upon addition of sucrose and seems to be regulated by multiple pathways (Shimotsu and Henner, 1986; Steinmetz et al., 1989). Several B. subtilis mutants that express LVS constitutively have been isolated and these mutations are located in three unlinked loci: (a)sacR which is a cis-regulatory region of sacB (structural gene for LVS) (Shimotsu and Henner, 1986); (b)sacS which codes for two genes, sacX and sac Y. Whereas sacX shares extensive homology to pts l ,

sacY is homologous to an Escherichia colt antiterminator protein BglG (Ayermich and Steinmetz, 1987; Debarbouilie et al., 1987; Steinmetz et al., 1988; Zukowski et al., 1988;) (c)ptsl which codes for phosphotransferase enzyme 1 (Crutz et al., 1990). In addition, the expression of sacB is also modulated by several unlinked genes such as degU and degQ that seem to affect most of the degradative enzymes in B. subtilis (Henner et al., 1988). The sacB gene from B. amyloliquefaciens was isolated to study the interaction &the various B. subtilis gene products such as sacS, degU) degR, degQ and ptsl with a heterologous LVS regulatory region. We report in this paper the isolation of the sacB[BamV] gene and its expression in B. subtilis.

RESULTS .'~ND DISCUSSION

Correspondence to: Dr. V. Nagarajan, E228/Rm 310C, Du Pont Co., P.O. Box 80228, Wilmington, DE 19880-0228 (U.S.~,.), Tel. (3027695-3984; Fax (302)695-9 ! 83. Abbreviations: aa, amino acid(s); Ap, ampicillin; B., Bacillus; bp, base pair(s); kb, kilobase(s) or 1000 bp; LVS, levansucrase; nt, nucleotide(s): oligo, oligodeoxyribonucleotide; ORF, open reading frame; R, resistance/resistant; RBS, ribosome-binding site; sacB, gene encoding LVS; sacB[BamP], gene encoding LVS from B. amyloliquefaciens; sacR[BamP], regulatory region of sacB [BamP]; tsp, transcription start point; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990 Fl,~e~J~erScience Publishers B.V. (Biomedical Division)

(a) Isolation of the sacB[BamP] gene A partial EcoRl library of B. amyloliquefaciens ATCC23844 in ,~Zap (Stratagene) was screened with two oli8os, 5'-GACGTTGGACAGCTGGCCATTACAAAC and 5'-ATGAACGGCAAATGGTACCTG'FI'CACTGAC, which had been synthesized based on tile published B. subtilis sacB gene (Steinmetz et al., 1985). The two positive phage clones were converted to plasmids designated as

90

pBE300 and pBE301 using helper phage IRI. Comparison of EcoRl restriction analysis revealed that pBE300 contained a 1.5-kb insert and p~,E301 contained a 2.3-kb insert consisting of a 1.5-kb and 800-bp EcoRl fragments. The sacB oligo probe hybridized to the 1.5-kb fragment. The restriction map of pBE301 is shown in Fig. 1.

E

0

~

1 E

E

0

I

1 H

P

~

,.I H E

sacB[BamP] Fig. 1. R e s t r i c t i o n m a p o f p l a s m i d pBE301 containingsacB [BamP].T h i n line,vector;blackenedbox;s,::cB [Bame] ; z i g - z a g l i n e , B . amyloliquefaciens

DNA.B,BamHI;E, EcoRI, H,HindlII;M,Smal;P,PstI, gN,EcoRV;X, Xba.

(b) Nucleotide sequence determination Plasmid pBE301 was digested with various restriction enzymes and subcloned into both M13mpl8 and M13mpl9. Several independent and overlapping clones were sequenced resulting in a contigue of 2350 bp (Fig. 2A). Analysis of the nt sequence revealed the presence of a promoter sequence that can be recognised by B. subtilis RNA polymerase Ea A and a Gram + RBS (McLaughin etal., 1981; Moran etal., 1982). The nt sequence also suggested the presence of a stem-loop structure with a free energy of 24.5 kcal between the putative promoter and the RBS (Fig. 2B). Computer analysis of the nt sequence revealed the presence of a large ORF which coded for a mature protein containing 443 aa and a signal peptide coding for either 29 or 31 aa. However, only codon -29 had the correct spacing between the RBS and the start codon.

A GAATTCCTTCAGGAAAAGAACGATGGCTGTCTTATTAGCGGTTGCAGGcAcATTT&TTTTGGTcAcACAcGGGAATGTcGGCAGCCTGTcTATAT~CGGTCT •GCTGTTTTTTGGGGCATCAGCTCGGCATTTGCGCTGGcGTTTTA•AC•CTC•AGccG•ATCGG•TTTTGAAGAAATGGGGcTc•Gc•ATTATTGTcGGATGGGG•ATGCTGATGCGGAG CCGTTCTCAGCcTGATT~AGC~GC~TTGGAAGTTTGAAGGCCAATGGTCGTTGTCcG~ATATGc~GcGAT~GTGTTTATcATcATTTT~GGAA~GcTcATcGCTTTTTATTGCTATTTGG AAAGCCTGAAATATCTGAGTGccTcTGAAA•CAG••TccTcGccTGTG•AGAG•cG•TGTcAGcAGcTTTTTTAGCGGTGATcTGG•TGcATGTTcccTTcGGAATATcAGAATGG•TGG ~TACTTTA~TGATTTTAGccA~ATCGCTTTATTAT~TATcAAGAAAAAATAA~cTcTcTTTTTTA~AGAGGTTTTTcccTAGGccTGAAG~AcccTTTAGTcTcAATTA~ccATAAATT AAAAGGCcTTTTTTCGTTTTAcTAT~ATT~AAAAGAGGAAAATAGAccAGTTGTcAATA~AATcAGAGTcTAATAGAATGAGGTcGAAAAGTAAATcAcG~AGGATTGTTAcTGATAAAG CAG~CAAGA~CTAAAATGTGTTAAGGG~AAAGTGTATTcTTTGGcGTcATc~TTAcATATTTTGGGTcTTTTTTT~TGTAAcAAAc~TGccATCcATGAATTcGGGAGGAT~GAAACGG CAGATCGCAAAAAACAGTACATACAGAAGGAGACATGAAC ATG AAC ATC AAA AAA ATT GTA AAA CAA GCC ACA GTT CTG 901 me~ ash ~le lyS lys ile val lys gln ale ~hu val leu -29 ACT TTT ACG ACT GCA CTT CTG GCA GGA GGA GCG ACT CAA GCC TTC GCG AAA GAA AAT AAC ~h~ phe ~hr ~hr ale leu leu ala gly gly ale thr gln ale phe ale lys ~lu asn ash -1

+1

CAA gln

AAA lys

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GTC val

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CTG leu

CAG gln

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AAA iys

CAG gln

C~G gln

CAA gln

AAC ash

GAA glu

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CAA gln

GTG val

CCT pro

CAA gln

TTC phe

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TCA see

ACG th~

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~AT a~n

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GAG glu

TCT ~eE

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CTT leu

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GTG val

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CAA gln

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GCT ala

GCA ale 50 GAA glu

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CAC his

GTT val

GTG val

TTT phe

GCT ala

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GCG ale

GGA gly

AGC set

CCG pro

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GAC asp

GCT ale

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GAC asp

ACA ~hr

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GAC a3p

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GGT gly

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^~ lys

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AAA lys

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342 582 822

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1141

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100

1381

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TAACAGCAAAAAGAAAATGCCGATACTTCATTGGCATTTTCTTTTATTTCTC~CAAGATGGTGAATTC 2350

B 710

720

730

u

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-

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AAGACCUAAAAUGUGU AAGGG

GUGU

UUCUGGGUUUUAUACA

UGCG

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770

760

UUCCC -

-UAC

C U GUU

750

Fig. 2. The nt sequence ofsacB[BamP]gene and second,-,rystructureofsacR[BamP].(A) The sacBgene. FlasmidpBE301 (see Fig. 1) was digested with various restrictionenzymes and various fragmentswere subclonedinto Ml3mpl8 and M13mpl9 (Maniatis et al., 1982). Single-strandedDNA fromthe phageswereisolatedand the nt sequencewas determinedusinga Sequenasesequencingkit (United States BiochemicalCorp.,Cleveland,OH). Both strands have been sequencedusingoverlappingclones.The sequencehas beensubmittedto the EMBLdatalibraryunderaccessionnumberX52988. (B) Structureof sacR[BamP].The secondarystructureo[the putativeregulato,ryregion(nt sequence708-774) was generatedby computeranalysisusingFold programof sequence analysissoftwarepackage(Genetics ComputerGrou,p ofthe Universityof Wisconsin). Numbersreferto those in partA.

(c) Comparison of the nt sequence of sacB[BamP] with that of Bacillus subtilis sacB Shimotsu ~nd H ~ n e r (1986) have de~ermined the tsp of B. subtilis sacB. Comparison of the nt sequence from -55 to + 115 ( + 1 denotes the tsp for B. subtilis sacB) shows 90 % identity with extensive homology at both the promoter and regulatory region (Fig. "~). The co~ing region also showed 90% identity; 4% were conservative changes and 6% of the residues were different (data not shown). A small ORF has been described in the case of B. subtilis in the sacB region. However, this ORF is not translated in vivo

(Shimotsu and Henner, 1986). a similar ORF is absent in the case of sacR [BamP]. Henner et al. (1988) have identifled an upstream activating sequence for B. subtilis sacB as

the possible site of action for degU anddegQgeneproducts. Comparison of the upstream nt sequences from -56 to -263 showed only a 25 % homology. (d) Sucrose inducible expression of sacB[BamP] The sacB[BamP] gene from pBE301 was cloned into an E. coli-B, subtilis shuttle phagemid vector pB E20 (HindIII-

digested pTZ18RligatedwithHindIII-digestedpC194)re-

92 -263

1 ......

40 s a c B - B , subtili$

GATCCTTTTTAACCCATCA . . . . CATATACCTGCCGTTCACTAT I

IIIIII

I

I

I

I

I

I

I

II

III

II

399 C~G~CAGCA~CTTTTTTAGCGGTGATCTGGCTGCATGTTCCCTTCGGAAT 448

sacB[BarnP]

41 TATTTAGTG;~J%ATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAG 90 i l II Ill I i II i l 449 ATCAG~TGGCTGGGT~CT~TACT~ATTTTAGCCACCATCGCTTTATTA.

497

91 GCAACTTTATGCCCATGCAACAG~,%CTATAAAAAATACAGAG~-TG;~ 140 I

I

II

~'

I

I II

l

498 .....TCTATC~.G~))$~,TAACCTCTCTTTTTTAGAGAGGTTTT~CCC 542 141AGAAACAGA~'AGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCA~TCCTT 190 I lllll Ill l II Illl 543 TAGGCCTGAAGCACCCTTTAGTCTCAATTACCCATAAATTAAAAGGCCTT 592 .-55

.

-35

191TTAT.GATTTTCTATCAAACAAAAGAGGAAAATAGACCAG~~TCCA II I I Ill llllll lllIIllllllllllllllll!lll II 593 ~ C G ~ C ~ C ~ C ~ G ~ G G ~ U ~ G ~ C C ~ C ~ G T C ~ T ~ G • -10___. +1 . . . 240 AACGAGAGTCT~~.TGAGGTCGAAAAGTAAATCGCGCGGGTTTG,T II lllIIllll}lIIIIlilllllllllllllIIlll Ill II lllll 643 AATCAGAGTCTAA~AGAAT~GAGGTCG_AAAAGTAAATCACGCAGGATTGTT -

239 6~2

289 692

sacR~

290 ACTGATAAAGCAGGC~AGA6CTAAAATGTGTAAAGGGCAAAGTGT~TACT 339 Illlllllllllllllllllllllllllllll

Illllllllllll~l

II

693 ACTGATAAAGCAGGC~.AGACCTAAAATGTGTTAAGGGCAAAGTGT~TTCT 742 340[TTGGCGTCACCC-CTTACATATTTT-AGGTCTT]TTTTTATTGTGCGTAACTA 389 ]llii!llll llllllllllllll llllllllllll I llIIl l 743 [TTGGCGTCATCCCTTACATATTTTGGGTCTT~TTTTCT .....GTAACAA 787 390 ACTTGCCATICTTCAAACAGGAGGGCTGG;%AGAAGCAGACCGC.TAACAC 437 II II I I I II II II I Ill Ill III II II ~

~CC~GCC~CC~G~CGG~G~CG~CGGC~C~C~C

a~

438 A G T A C A T ~ G G A G A ~ A T G A A C ~ C A T C A A ~ G T T T G C A ~ ]lllllll

! ~!l!!lll~llil;I

~l[~;f:llllllll

487 Ill

Illl

838 A~TACATAC~GI~O~GGAGA~;~YGAAC.[ATt~ACATC~O.AAAA~GTAAAA 886 •

RBS

488 CAAGCAACAGTAT 500 lllll I Ill 887 CAAGCCACAGTTC 899

f

met

Fig, 3. Comparison of the B. s.bti~-sacB sequence with sacB [BamP]. Nucleotide sequences of the promoter and upstream sequences of8. sub~iliz-sac8(top lines) and sacB [BamP] (bottom lines) are compared. + I denotes the tsp ~ r the B. subtilissacBtranscription, as determined by Shimotsu and Henner (1986).

suiting in pBE501, pBE504 is similar to pBE501 except it contains two additional restriction-enzyme recognition sequences in the signal peptide coding region and these changes do not alter the aa sequence of the signal peptide. B. subtilis strain BG4103[pBE504] was grown in medium B to an A6oo,m -- 0.5 and varying concentrations of sucrose (100/~M-100 mM) was added and 2 h lt°er the extracellular LVS activity was measured. The cell de~,sity of the various ,.ultures was comparable but the amount ,~f LVS activity varied with the amount, of sucrose added (Fig. 4). The uninduced culture had an activity of < 0. I0 units while a maximal activity of 2.'. units was observed when the initial sucrose concentration was 4 raM. However, with increasing sucrose concentration LVS activity decreased and at the maximal sucrose concentration tested (I00 mM) an activity of 0.80 units was observed. The concentration of sucrose needed to maximally induce B. subtilis sacB has been reported to be 30 mM sucrose (Lepesant et al.. 1976; Steinmetz eta!., 1989).

The sucrose induction of sacB[BamP] on a multicopy plasmid in B. subtilis suggests that B. subtilis-SacY probably can act as an antiterminator and interact with sacR[BamP]. Our results show that 4 mM sucrose was sufficient to maximally induce sacB[BamP] in B. subtilis. The presence of the multiple copies of the sacR[BamP] might titrate some of the factors and thus we expected to find a lower concentration of sucrose to induce the sacB[ RomP]. However, we did not expect to see a decrease in the amount of LVS accam, dqted when induced with high concentrations of s,lcrose. Whether this observed decrease in the accumulated LVS is due to sacR[BamP], gene dosage or growth medium is not clear. The regulation of sacB in B. subtilis seems to be similar to the regulation of the bgl operon of E. coli (Mahadevan et ~., 1937; Schnetz et al., 1987; Steinmetz et al., 1988). Amstar-Choder et al. (1989) have shown that BglG which is homologous to SacY, acts as an antiterminator when dephosphorylated. The phosphorylation of BglG is modu-

93

2.0

5 ~:

1.0

,z.,

0.1

I

I_

1

10

100

SUCROSE [mM]

Fig. 4. Expression of sacB [BamP] in B. subtilis. B. subtilis strain BG4103 (3sacB, trpC2) containing pBES04 was grown in medium B (per liter: tryptone 33 g/yeast extract 20 g/NaCi 7.4 g/Na,HPO 4 8 g/KH:PO4 4 g/casamino acids 20 g/glycerol 60/~M/MnCI: 0.06 mM/FeCI3 S0~ nM/NaOH to pH 7.57. LVS was partially purified from 10 ml culture supernatant by ethanol precipitation and resuspended in 500 #! of 50 mM K. phosphate buffer pH 6.0 containing 20% glycerol (Dedonder, 19667.50/~! ofthe partially purified LVS was preincubated in 50 mM K. phosphate buffer (850 pl) at 37 ° C for 5 rain. The reaction was initiated by the addition 100 #! of sucrose (400 raM). Samples were withdrawn at 0,15 and 30 min and LVS was inactivated by incubation at 70°C for 5 rain. The amount ofgiucose present at 0, ~5 and 30 rain was determined using Glucose Trindcr reagent from Sigma. O - ~ un~, of LVS activity is defined as 1/~g ofglucose released/min/ml of the culture supernatant.

lated by the protein kinase action of BglF. Regulation of sacB may be more complex than the E. coil bgl operon because Steinmetz et al. (I 989) observed a low level ofsacB induction in strains carrying a deletion in sac Y. In addition, ptsl mutants of B. subtili~' express sacB constitutively (Crutz et al., 1990). Thus, the availability of a second LVSencoding gene from another Bacillus species should aid in studies towards understanding the complex mechanism/s involved in the sucrose regulation of sacB in B. subtilis.

ACKNOWLEDGEM ENTS

We thank Dennis Henner for providing strain BG4103 and Georges Rapoport, Mark Zukowski, Ethel N. Jackson and Mark Payne for useful discussions and Helene Albertson for excellent technical assistance.

REFERENCES Amster-Choder, 0., Houman, F. and Wright, A.: Protein phosphorylation regulates transcription of the ~-glucoside utilization operon in E. coll. Cell 58 (1989) 847-855. Ayecmich, S. and Steinmetz, M: Cloning and preliminary characteriza-

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