A Derepression System Based on the Bacillus subtilis Sporulation Pathway Offers Dynamic Control of Heterologous Gene Expression

By rewiring the sporulation gene-regulatory network of Bacillussubtilis, we generated a novel expression system relying onderepression. The gene of interest is placed under the controlof the abrB promoter, which is active only when Spo0A is absent,and Spo0A is controlled via an IPTG (isopropyl-ß-D-thiogalactopyranoside)-induciblepromoter.

INTRODUCTION

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Introduction

We designed a novel system to express and secrete (heterologous)proteins in Bacillus subtilis. Instead of using an inducer toactivate protein production, we used a derepression system.This allows growth of the host to high cell densities in thepresence of the inducer. Upon removal of the inducer, high expressionlevels are obtained. To generate such a system, we made useof the well-studied sporulation phosphorelay of B. subtilis.

Controlled derepression of abrB by relief of Spo0A.

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Controlled derepression of abrb...

As an adaptive ability in response to starvation, B. subtilisis able to form highly resistant endospores (13). The processof sporulation is governed by a multicomponent phosphorelay(11). Multiple environmental and physiological signals are fedinto this system, and under appropriate conditions this leadsto phosphorylation of Spo0A (Spo0A $~$ P), the key sporulation transcriptionfactor (1). A major role of Spo0A $~$ P is to repress abrB expression(9, 10).

In the absence of Spo0A $~$ P, abrB gene expression is constitutivelyhigh (12). To test whether we could use this feature of thesporulation pathway to construct a derepression system to expressheterologous proteins in B. subtilis, we cloned the gene encodinggreen fluorescent protein (GFP) behind the abrB promoter (Fig.1). In the resulting strain (17), cells highly express GFP duringexponential growth, and fluorescence is reduced upon entry intothe stationary growth phase (data not shown). Ireton et al.have shown that the abrB promoter is repressed by artificialinduction of Spo0A-Sad67 (herein called Spo0A*), a constitutivelyactive variant of Spo0A (3). Knowing this, we introduced theIPTG (isopropyl-ß-D-thiogalactopyranoside)-inducibleSpo0A* construct into our P_abrB-gfp strain, named A-gfp. Allbacterial strains and plasmids used in this study are listedin Table 1. Bacteria were grown and transformed using standardtechniques.

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FIG. 1. Sporulation-based derepression system. A simplified schematic representation of the regulatory network used in this study is shown. Perpendicular symbols and arrows represent negative and positive actions, respectively. 0A* represents P_spac-spo0A-sad67. High levels of IPTG induce Spo0A*, which represses P_abrB. AbrB and the protein of interest are not produced, and there is no repression of the extracellular proteases. If IPTG is absent, Spo0A* is not produced and P_abrB is active, yielding high levels of AbrB and the protein of interest, while AbrB concomitantly represses the extracellular proteases.

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TABLE 1. Bacterial strains and plasmids

Flow cytometric measurement of GFP fluorescence.

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Flow cytometric measurement of...

Cells were grown overnight in TY medium (16) containing 100µM IPTG and diluted 30-fold to an optical density at 600nm (OD₆₀₀) of 0.05. At an OD₆₀₀ of $~$ 1.0, cells were diluted 10-foldin minimal medium, and a 1-ml suspension (without glass beads)was treated with a Mini-Bead-Beater-8 (Biospec Products) for1 min at maximal speed to separate cell chains into individualcells. Two hours later (end of log phase), another sample wasmeasured. GFP fluorescence was measured using a Coulter EpicsXL-MCL flow cytometer (Beckman Coulter). The average fluorescenceof 20,000 cells was determined using WinMDI 2.8 (http://facs.scripps.edu/software.html)and plotted against IPTG concentrations (Fig. 2). As shown inFig. 2, GFP expression under the control of P_abrB is high withoutinducer but is strongly reduced upon increases in levels ofSpo0A*. When the native spo0A gene is deleted, GFP expressionis further increased. The maximum concentration of GFP in thisstrain was quantified by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) relative to bovine serum albuminstandards. Dry-matter concentrations of biomass were calculatedusing a predetermined correlation factor of 0.33 g (dry weight)of cells per OD₆₀₀ unit (19). The concentration of GFP withan average fluorescence per cell of 550 arbitrary units was16.7 mg GFP/g (dry weight). Interestingly, under inducing conditions(in the presence of Spo0A*), P_abrB is more tightly repressedin the spo0A ( ${Delta}$ spo0A) mutant. In the wild-type strain, the abrBpromoter is still leaky and shows optimal derepression of abrBwhen Spo0A* is induced with 50 µM IPTG.

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FIG. 2. Controlled activation of P_abrB-gfp via derepression. Expression of Spo0A* was induced with various concentrations of IPTG. P_abrB expression was measured during mid-exponential growth (A) and late exponential growth (B) using a P_abrB-gfp fusion. Expression of P_abrB-gfp in the presence and absence of endogenous spo0A was measured (strains 0A*/A-gfp and 0A*/ ${Delta}$ 0A/A-gfp). The units for GFP fluorescence are arbitrary units (AU) and are identical in panels A and B.

Secretion of Clostridium perfringens ß-toxoid.

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Secretion of clostridium...

To examine whether the system also enables the controlled expressionof a secreted heterologous protein, we placed the gene encodingthe C. perfringens ß-toxin (cpb) behind the abrB promoterand combined this construct with the strain carrying the spo0Amutation ( ${Delta}$ spo0A) and the inducible Spo0A*.

The full coding sequence for ß-toxin (cpb) was amplifiedby PCR with plasmid pXB10 as a template. C. perfringens ß-toxinis a secreted protein with a Sec-type signal sequence and isan important component in animal vaccines against C. perfringenstypes B and C (7).

To visualize ß-toxin secretion, total medium proteinswere 10x concentrated by trichloroacetic acid precipitationand separated by SDS-PAGE as described previously (14). ß-Toxinwas detected using Western blotting as described previously(7). As shown in Fig. 3A, at high IPTG induction levels, noß-toxin could be detected. Upon derepression fromSpo0A*, ß-toxin accumulated in the growth medium.These results show the versatility of the derepression systemand demonstrate that (heterologous) gene expression can be accuratelycontrolled.

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FIG. 3. Controlled secretion of ß-toxin via derepression. abrB derepression results in reduced extracellular proteolytic activity. (A) Detection of 35-kDa ß-toxin secreted by strain 0A*/ ${Delta}$ 0A/A-ß (P_abrB-cpb P_spac-spo0A* ${Delta}$ spo0A). (B) Detection of secreted 35-kDa ß-toxin by strains 0A*/ ${Delta}$ 0A/A-ß (P_abrB-cpb P_spac-spo0A* ${Delta}$ spo0A) and A-ß (P_abrB-cpb). Time (T) zero is directly after resuspension in fresh medium. The increased background in later time points for 0A*/ ${Delta}$ 0A/A-ß is caused by cross-reaction of the ß-toxin antibody with some intracellular proteins released into the medium by cell lysis. (C) Extracellular proteolytic activity of the cultures depicted in panel B.

To examine whether the described derepression system can beactivated in cultures with a high cellular density, which isrequired when producing toxic products, we grew strains A-ß(P_abrB-cpb) and A-ß/0A*/ ${Delta}$ 0A (P_abrB-cpb P_spac-spo0A* ${Delta}$ spo0A) to dense cultures in TY medium containing 250 µMIPTG (full repression). Next, cells were spun down, washed once,and resuspended in fresh TY medium without IPTG. Medium fractionswere collected at timely intervals and assayed for ß-toxinsecretion. As shown in Fig. 3B, within 20 min after resuspension,P_abrB was derepressed in A-ß/0A*/ ${Delta}$ 0A and ß-toxincould be detected in the growth medium. ß-Toxin continuedto accumulate in the medium up to 2 h after derepression inthis dense culture, and we were able to recover ß-toxinuntil 5.5 h after suspension. In the presence of a functionalspo0A gene, however, secreted protein could not be observedafter 3.5 h.

abrB derepression results in reduced extracellular proteolytic activity.

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abrB derepression results in...

Our results show that secreted ß-toxin is more activelydegraded in the presence of a functional spo0A gene. Many ofthe known extracellular proteases are under either the director indirect control of Spo0A (6). To examine whether the increaseddegradation of ß-toxin is related to increased extracellularproteolytic activity, we assayed the growth medium for proteolyticactivity. Total protease activity was measured using the Rocheresorufin-labeled universal protease substrate as describedby the manufacturer. Direct optical readout of the OD₅₇₄ wasplotted. As depicted in Fig. 3C, only a minor proteolytic activitywas measured in the ${Delta}$ spo0A strain. In the presence of spo0A,strong protease activity was observed starting after 150 minof resuspension in fresh medium, when cells enter stationarygrowth phase (data not shown).

Induction and deletion of spo0A.

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Induction and Deletion of...

The use of sporulation-deficient mutants (such as the ${Delta}$ spo0Astrain) is common practice for large-scale high-density fermentationprocesses, mainly because it prevents the formation of spores,which are hard to remove from the growth system (8). Strainstability is not an issue for these mutants, since sporulationis purely an adaptive phenotype and not essential. The additionof IPTG in the case of our Spo0A* strain led to induction ofSpo0A, and therefore undesirable sporulation could occur morerapidly. However, we did not observe sporulation under our cultureconditions. The process of sporulation is tightly controlled,and when only spo0A is induced during logarithmic growth, notall essential components for sporulation are expressed, preventingpremature sporulation of the culture (2).

Concluding remarks.

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Concluding remarks.

In this paper we show how a naturally occurring gene-regulatorynetwork can be adapted to serve as a tailored expression system.An additional benefit of using this endogenous pathway is thatthe system is based on self-cloning via homologous recombination,keeping the introduced foreign DNA to a minimum. Furthermore,upon activation of the abrB promoter, extracellular proteolyticactivity is reduced.

ACKNOWLEDGMENTS

R.N. was supported by Intervet International B.V. (Boxmeer,The Netherlands). J.-W.V. was supported by grant ABC-5587 fromThe Netherlands Organization of Scientific Research TechnologyFoundation (NWO-STW).

FOOTNOTES

* Corresponding author. Mailing address: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31 50 3632093. Fax: 31 50 3632348. E-mail: o.p.kuipers{at}rug.nl.

Published ahead of print on 9 February 2007.

Both authors contributed equally.

REFERENCES

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