uene. y:b (l~yU) IJl-141 Elsevier

137

GENE 0376 !

Efficiency o f t r a n s c r i p t i o n a l t e r m i n a t o r s in B a c i l l u s s u b t i l i s (Recombinant DNA; exoenzymes; gene expression; transcriptional termination; transcriptional interference; reporter gene)

Gerard F. Hess and R. Scott Graham Molecular Biology Research. The Upjohn Company. Kalamazoo. MI 49007 (U.S.A.) Received by R.E. Yasbin: 26 April 1990 Revised: 2 July 1990 Accepted: 24 July 1990

SUMMARY

To promote more efficient synthesis of heterologous gene products in a Bacillus subtilis host, we have developed a system for rapidly testing the effect of a putative terminator on in vivo gene expression. Terminator structures from the Bacillus amyloliquefaciens amyE gene, the Bacillus licheniformis penP gene, the B. subtilis bgIS gene, and the Bacillus thuringiensis cry gene were subeloned and inserted into a vector in such a way as to disrupt expression of the cat-86 gene. Comparisons are made between gene expression levels and the stabilities of the respective stem-loop structures.

INTRODUCTION

A number of factors determine the efficiency at which a heterologous gene product may be produced by a microbial expression host. These factors include distinct but interrelated conditions such as stable inheritance of the target gene, 8ene dosage, strength of the transcriptional promoter, the efficiency with which the transcribed message is translated, and the stability of the transcript. The process of transcriptional termination has been indicated in playing a role in a number of these processes. An important manner in which termination influences the Correspondence to: Dr. G.F. Hess, Molecular Biology Research, 7242/267/5, The Upjolm Company, 301 Henrietta Street, Kalamazoo, MI 49007 (U.S.A.) Tel. (616)385-5303; Fax (616)385-7373.

Abbreviations: A, absorbance (! cm); amyE, gene encoding u-amylase; bp, base pair(s); bgl$, gene encoding ~-glucanase; CAT, Cm acetyltransferase; cat.86, gene encoding CAT from B. pumilus; Cm, chloramphcnicol; cry, gene encoding crystal toxin protein; ExoilI, exonuclease lIl; kb, kilobase(s) or 1000 bp; Kin, kanamycin; LB, Luria-Bertani (medium); nt, nueleotide(s); oligo, oligodeoxyribonucleotide; penP, gene encoding ~.lactamase; Pollk, Klenow (large) fragment of E. coil DNA polymerase I; RBS, ribosome-binding site; SPO2, B. subtilis bacteriophage SPO2; tic, thin-layer chromatography; UWGCG, University of Wisconsin Genetics Computer Group. 0378-1119/90/$03.50 © 1990 Elsevier Science PubfishersB.V.(BiomedicalDivision)

expression of heterologous genes is in the stabilization of the message. Stable stem-loop structures associated with termination structures in Escherichia coli have been shown to prolong message half-life through discouraging ribonuclease activities (Guarneros et al., 1982). A similar result has been described in B. subtilis exerted by the termination structure derived from the B. thu~nglensis crystal toxin protein gene (Wong et al., 1983). The messenger RNAs of heterologous constructions containing this termination structure have been shown to be more stable than the transcript without the structure (as determined by RNA half-life measurement). Thus, host strains carrying the construction containing the terminator yielded higher steadystate levels of message, enhancing the level of gene expression (Wong and Chang, 1986). To promote more rapid expression of heterologous gene products in a B. subtilis host, the in vivo effect of terminator structures on the expression of a downstream reporter gene (cat-86) was examined. The data were employed to determine whether a correlation could be made between the predictive stability ofthe stem-loop component ofthe terminating structure and the overall level of cat-86 gene expression. It was hoped that this would lead to predictive criteria and a rapid in vivo screen for structures which function as transcriptional terminators and which promote transcript

138 stability as has been observed for the crystal toxin terminator.

EXPERIMENTAL AND DISCUSSION

(a) Plasmids Four putative terminator structures, describing a hierarchy of stem-loop stab'titles, were subcloned from axeenzyme genes isolated from Gram + organisms. The exoenzyme genes were: (i ) amyE, encoding B. amyloliquefaciens ~-amylase (Lehtovaara et al., 1984; Takkinen et al., 1983), (ii) pen/', encoding B. licheniformis /~-lactamase (Neugebauer etal., 1981), (iii)bglS, encoding the B. subtilis /~-glucanase (Murphy et al., 1984), and (iv)cry, the gene encoding the B. thuringiensis crystal toxin protein (Wong et al., 1983). The crystal toxin protein terminator was constructed in vitro utifizing synthetic oligos homologous to published sequence data (see Wong et al., 1986). To assay the efficiency of termination two vectors (pPL703 and pPLT08; Duvall et ai., 1983) were used. Both vectors contain a multilinker region proximal to the RBS and.structural gene for the cat-86 gene on a Gram + compatible replicon. This configuration allows for the insertion of a putative transcriptional terminator into the linker region, between the transcriptional promoter and the RBS, and the subsequent comparison of the resulting enzyme expression level with expression levels of the appropriate varent constructions (Table I and Fig. 1).

(5) Terminators (1) o¢-Amylase terminator (troy) The B. amyloliquefaclens 0c-amylaseterminator contains a palindromic sequence which can form a stable stem.loop (AG = -23.1 kcal/mol, Fig. 2). To the 3' side of the stem, beyond the reported terminal nt (Lehtovaara et ai., 1984), there is a 10-nt long suetch of U's, interrupted by one A residue (Fig. 2; Lehtovaara et al., 1984; Takkinen, 1983). This terminator structure is symmetrical (Lehtovaara et ai., 1984), and consequently may function in terminating transcription in both directions.

TABLE I Plasmids and genetic constructions (A) Source plasmids for exoenzyme genes and assay vectors a pPLT03 Bacffus Genetic Stock Center pPL708 Bacillus Genetic Stock Center pSG402 /~-Glucanasefrom B. subtilis BRISl pAmylo202 u-Amylase from B. amyloliquefaciens H pPenPe24 /~-Lactamasefrom B. lichen~'ormis (B) Strategies for genetic constructions b (c) SP02ixomoter(P~ pAmylo202 I

~

416~o

~

pAmYb202 L " - ' ~ Ha°Ill"BamH' 416bp ~

oUOAMTERM . ~

BwwHI - PStl

63bp OSG402 ~.~

__

pAM.1708-1

Pt pAMREV5.1 708

I~lrt~HI + Hindl

Ball - Pvuil

710bp

(e) O~os [~,,.~

1~4M703-10

BamH,+ Pstl pUCAMTERMREV~ pAMREYS.1703

pPENPe24 ~=~

Pt t

BamHI ÷ PMI Haelll - BamHI

v

-~

pUCBLATERM

~

pBLATO3-1

8anfrll. PsrI pUCGRYTERM ~ pCRY270~.2

~

pBLA7QB-8

Pr ~ ~ pCRYT08-4 Pr

E(~RI + PM I

,~uQA 210bp

~- pUCGLUGTERM~

pGLUCTO~.9

~

pQLUC708-401 Pt

I~-]b 1Ec°RI'MIJ°I' 9~ ~

pOLUT3.S

~1)

pGLUT3-5708

" B, subtills BRI 51 was utilized as the host fo~"expression of all genetic constructions, b Terminator sequences were subcloned first into an £. coll compatible vector (a) to allow for introduction of useful restriction sites, and subHquantly into plasmid pPLT03 (h) Liption of DNA fragments with incompatible cohesive ends was achieved through the blunting of the appropriate ends utilizing Pollk, and then performing the ligation reaction under conditions fuvodng blunt.end ligation. The 284.bp EcoRl fragment containing the SPO2 promoter was isolated from pPLT08, and was ligated into the £coRl site of each of the terminator.pPLT03 con. structs (e) The promoter orientation ofeach construction was determined through restriction analysis and nt sequence analysis either by the method of Maxam and Gilbert (1980), or by the dideoxy chain-termination method of ganger etal. (1977). (d) Deletion of specific nt sequences was achieved through use of an Exolll-S ! nucleue kit CErasea-base' system; Promege). (e)The to~ was constructed in vice utilizing synthetic oligos homologous to published sequence data (see Wens et el., 1986, and Fig. 2).

$ |

od KmR

~ aPe2 Pr

RBS

cat.aS

Fig. 1. Schematic diagram of the physical map of pPL708 vector, Plasmid I)PLT08 was constructed from pPL703 through addition of a 284-bp £coRl fragment containing a bacteriophage SPO2-derived transcriptional promoter, oriented to direct expression through the linker region into the cat.86 gone (Duvall et el,, 1983).

139

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(c)

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kcal/mol: -23.1

kcal/mol: -15.0

kcal/mol: -22.6

kcallmol: -16.8

Fig. 2. Predicted m R N A secondary structures and stabilities of putative termination structures. Structures were generated using the Fold program designed by Zuker a n d Stiegler (1981) as included in the U W G C G Genetic sequence analysis soflworepackage, version 5.1 (Devereux et al., 1984). The structures were drawn through use o f the Squiggles program of Osterburg and S o m m e r (1981), as included in the U W G C G package. Default settings for rules o f folding and b o n d energies were those o f Frier et al. (1986) and the values are in kcal/mol. Sequence s h o w n includes only that immediate to

the putative terminationstructure.Terminatorfrom:(a) B. amyloiiquefaclensor-amylase(to,~); (b)B. lichen~formisp-lectamasc(t,~); (e) B. zhuringenesis crystal toxin protein(to,y);(d) B. subtgis/~-glucanase(tbsz).

This hypothesis was tested by subcloning the 416-bp fragment containing the terminator into the marker plasmids in both the native 5'- to -3' orientation, as well as in the reverse, 3'- to -5' orientation (Table I). When the terminator was in the reverse orientation (pAMREV 708), cat-86 expression was reduced by 93~o (Table ll) suggesting efficient termination. In contrast, when the terminator structure was in its native orientation (pAMT08-1), cat-86 expression was approx. 150~o of the parent strain (Table If). Within the nt sequences 3' to the region of dyad symmetry (bp 1730-1780; see Takkinen et ai., 1983) are sequences (bp 1870-1900) which closely resemble the -35 and -10 consensus sequence (TTGACA and TATAAT, respectively) recognized by the ¢43 RNA polymerase orB. subtilis (Moran et ai., 1982). Placement of the terminator-promoter-containing fragment in the marker plasmid in the terminator's native orientation could direct the expression of the cat-86 gene from the putative downs)ream promoter. Reversal of orientation could result in steric hindrance between polymerase molecules proceeding in opposite directions, resulting in reduced cat-86 expression.

The putative downstream promoter of the terminator fragment was removed by a two-step process of digestion with ExoIIl, followed by digestion with S l-nucleasegenerating plasmids pA2 703-1 and pA2-1 708-9 (Table I). Nonetheless, again CAT enzyme activity levels were significantly higher than those found in parent strains (Table II). The unexpected promoter-like activity associated with the amylase terminator fragment represents an anomaly which bears further investigation. S l-nuclease experiments described earlier (Lehtovaara et ai., 1984) locate the end of the in vivo transcript and indicate the sequence target.ed in this study as the putative terminator. As a conceivable explanation of these observations, the amylase terminator stem-loop structure, situated 5' to the translational start point of those transcripts which read through to the structurai gene, may lend considerable stability to the full-length transcript. The increase in message half-life would account for the unexpectedly high level of marker gene expression. Examination of this model is ongoing. (2) ~-Lactamase terminator (tap) The terminator from B. licheniformis

/~-lactamase

140 TABLE 11 Specific activity, and Yo efficiency of termination ~ Plasmid b

pPLT03 pAM703-10 pAMREV703 pA2703-1 pBLA703.1 pCRY2 703-2 pGLU703-9 pGLUT3.5 703

activity©

% Efficiency of terminationd

0.127 0.186 0.01 ! 0.194 0.130 0.142 0.242 0.129

-47.04 91.07 -53.35 -2.77 -12.57 -91.54 -9.56

Specific

Piasmid u

Specific activityc

% Efficiency of termination d

pGLU708.401 pAM708-1 pAMREV708 pA2-1708-9 pBLA708-8 pCRY2 708-4 pGLU708-401 pGLUT3.5 708

2.185 3.298 0.144 4.451 1.004 i.064 1.226 0.960

-50.94 93.40 -103.7 54.05 51.30 43.91 56.07

a Cultures were grown at 37°C, with aeration, in LB medium (Miller, 1972) with 0.2~o glucose, 10 ~g Km/ml (included as selection for the vector) and 0.5/AgCm/ml (included to obviate possible terminator activities afforded by the endogenous 5' stem-loop structure of the cat~86 8ene; see Ambalos et al., 1985). Cells were harvested by ¢entrifngation, washed with one half volume ofprewarmed growth medium without Cm, and resuspended at !/10 original volume with 25 mM Tris. HCI pH 7.8/10 mM EDTA. Cell suspensions were disrupted with three, 3-min cycles of sonication (high setting, S0~o duty cycle, microprobe horn attachment, model VCS00 Ultrasonic cell disrupter; Sonies & Materials, Inc.). Lysatcs were heated and cleared as described by Crabb and Dixon (1987). Protein concentrations were determined by the method of Bradford (1976). Appropriate aliquots of cell lysate were diluted to a final volume of 50 #1 with 25 mM Tris. HCI pH 7.8/10 mM EDTA. To each of these was added 40/d H20/35/d 1 M Tris. HCI pH 7.8/20/tl 4 mM acetyI-CoA (Pharmacia)/5/d D-threo-(dichloroacetyl)-[t4C]Cm (54 mCi/mmol, Amersham). The reaction mix was incubated at 37°C for 10 min and extracted with 400/d ethyl acetate, The ethyl acetate phase was retained and lyophilized. The acetylated and nonacetylated reactants were separated by tic on a 250-/~m layer, Whatman PE SIL-G, with 95 : 5 chloroform: methanol. Autoradiographywas at room temperature, overnight. Both acetylated and nonacetylated reactants were scraped from the tic plates, and quantitated through scintillation. b See Table I. c Values reported represent the nmol of [ t4C]Cm acetylated/#g of cellular protein per 10 min at 37°C. e The ~ termination is calculated by dividing the CAT-specific activity (determined by the plasmid containing the terminator) by the CAT activity specified by the fullyexpressed cat,86 gene. This value is then subtracted from 100% to give % termination. The minus designation ( - ) indicates promoter activity present instead of terminator activity.

contained a palindromic sequence which may allow the formation of a 15-bp stem=loop (.40 =-15.0 kcal/mol; Fig. 2) followed downstream by a stretch of twelve U's interrupted separately by one (3 residue and one C residue (Neugebauer et al., 198I). The reduction in cat.86 expression due to the ~.lactamase terminator is approx. 54% with only a small amount of fortuitous promoter activity in evidence ('Fable II).

(3) cry gene terminator (to,y) The crystal toxin protein terminator, to,y, originally isolated from B. thuringiensis (Wong et al., 1983), has been previouslydescribed as a positive retroregulator of heterologous gene expression (Wong and Chang, 1986). The palindromic sequence can form a 17-bp stem-loop with a single mismatched residue (AG-~-22,6 kcal/mol, Fig. 2), the stem-loop containing a 4 nt-long stretch of U's on the 3' side of the stem. The unexpected result from the in vitro constructed terminator structure used in this study was the poor efficiencyof termination (approx. 51%; Table ll). As previously described (Wong and Chang, 1986), tc,y markedly enhances the stability of the RNA transcript of the penP gene in comparison with the native bla terminator. The results from this study indicate that this instance of positive retroregulation may not be directly attributed to

greater efficiency of termination. More likely, the greater stability of transcripts containing tc,y reflects the refractive nature of the generated hairpin to RNase attack.

(4) p.Olucanase terminator (tbg~) The fourth putative terminator subcloned for this study, that from the B. subtilis bgl gene (Murphy et al., 1984) resembles an attenuator, in that it consists of a series of weak stem-loop structures which may form and dissociate according to the steric interaction of a translating ribosome. The computer algorithm utilized in this study did not allow the giucanase terminator sequence to fold into the same

structures depicted previously (Fig. 2; Murphy et el., 1984). In this study, the upstream interaction of a translating ribosome has been removed, and the overall reduction in cat-gO expression was quite low (approx. 44%; Table II). However, plasmid pGlu703-9, constructed without the SPO2 promoter directing transcription of the marker gene, showed that the fragment subcloned as containing the putative terminator also acted as a fortuitous promoter, detracting from the validity of the value for % efficiency of termination. Subsequent removal of the putative downstream promoter sequences (plasmids pGlut3-5 703 and pOlut3-5 708; Table I) allowed for an enhanced~ efficiencyoftermination (approx. 56%; Table If). The computer-generated

141 structure, its calculated stability, and the efficiency of termination it affords, are consistent with the predicted structures, stabilities and levels of termination seen with the bla and cry terminators.

ACKNOWLEDGEMENTS

We thank A. McNab and S.T. Motley for technical assistance, N. Theriault, J. Carter, and S. Pulaski for oligo synthesis, D.R. Siemieniak for assistance and instruction with the VAX computer, J. Hammond for excellent word processing, and D. Court for helpful discussions.

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transcriptional control of bacteriophage ~ mt gone expression from a site distal to the gene. Proc, Natl. Acad. Sci. USA 79 (1982) 238-247. Oorman, C.M., Moffat, L.F. and Howard, B.H.: Recombinant gcnoma which express chloramphenicol acetyltransferasc in mammalian cells. Mol. Cell. Biol. 2 (1982) 1044-1051. Lehtovaara, P., Ulmanen, I. and Palva, I.: In vivo transcription initiation and termination sites of an a-amylase gene from Bacillus amylolique. fadens cloned in Bacillus subti~s. Gene 30 (1984) 1!-i6. Maxam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with basespecific chemical cleavages. Methods Enzymol. 65 (1980) 636-639. Moran Jr., C.P., Lang, N., LeGrice, S.FJ., Lee, G., Stephens, M., Sonenshcin, A.L, Pero, J. and Losick, It.: Nucleotide sequences that signal the initiation of transcription and translation in Bacillussublilis. Mol. Gen. Genet. 186 (1982) 339-346. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, p. 433. Murphy, N., McConnell, DJ. and Cantwell, B.A.: The DNA sequence of the gnne and genetic control sites for the excreted B. subr///senzyme ~-glucanase. Nucleic Acids Res. 12 (1984) 5355-5367. Neugebauer, K., Sprengel, R. and Schaller, H.: Penicillinase from B a ~ licbeniformis: nuclcotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram.positive bacterium. Nucleic Acids Res. 9 (1981) 2577-2588. Osterburg, G. and Sommer, R.: Computer support of DNA sequence analysis. Comp. Prngr. Biomed. 13 (1981) 101-109. Sanger, F., Nicklen, S. and Coalson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Takkinen, K., Peuersson, R.F., Kalkkinen, N., Palva, 1., Sodedund, H. and Kanriainen, L.: Amino acid sequence of ~,-amylasefrom Bacillus amyloliquefaciens deduced from the nuclcotide sequence ofthe cloned gene. J. Biol. Chem. 258 (1983) 1007-1013. Wong, H.C. and Chang, S.: Identification of a positive retroregulater that stabilizes mRNAs in bacteria. Proc. Nati. Acad. Sci. USA 83 (1986) 3233-3237. Wong, H.C., Schnepf, E. and Whiteley, H.R.: Transcriptional and translational start sites for the Bacillus tAuri~ensts crystal protein gene. J, Biol, Chem. 258 (1983) 1960-1967. Zuker, M. and Stiegler, P.: Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 9 (1981) 133-148.