Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH (2024)

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Abstract

1 Introduction

The Gram-positive bacterium Bacillus subtilis produces a variety of extracellular enzymes including six different proteases, an α-amylase, a levansucrase, several β-glucanases and at least two different lipolytic enzymes [5–8]. The corresponding genes underly a complex regulation (see [12,14]) with DegS-DegU being the major two-component system involved [23]. Additionally, cross-regulations were found with other two-component systems regulating sporulation (KinA-KinB/Spo0F-Spo0A) and natural competence (ComP-ComA/ComQ) [22]. Furthermore, several regulatory genes including sacV, senN, senS, tenA, tenI, sinR, sinI and abrB were shown to up- or down-regulate the production of extracellular enzymes in B. subtilis[14].

At present, the regulation of the extracellular lipolytic enzymes LipA and LipB is unknown. When the lipA gene was first isolated, DNA sequence comparisons of the potential promoter region indicated a σ^A-dependent constitutive expression [3]. First experiments on the lipase regulation in B. subtilis indicated a differential expression of both genes but the regulatory signals remained unknown [6]. In the present study the regulation of the lipase gene expression was investigated by first constructing the lipase deletion mutants B. subtilis TEB1010 (ΔlipA), TEB1020 (ΔlipB), and TEB1030 (ΔlipAΔlipB). We identified two environmental factors, namely external pH and amino acid supply, which are responsible for the differential expression of LipA and LipB in B. subtilis.

2 Materials and methods

2.1 Bacterial strains, plasmids, media and growth conditions

The bacterial strains used in this study are listed in Table 1. LB medium was used as the rich medium for both B. subtilis and Escherichia coli. In some experiments the LB medium was supplemented with glucose to a final concentration of 1% (w/v). Buffered minimal medium (Spizizen minimal medium (SMM): 125 mM K-PO₄ buffer pH 7, 15 mM (NH₄)₂SO₄, 3.4 mM Na-citrate, 0.8 mM MgSO₄, 1% (w/v) glucose) was used as the chemically defined growth medium. Histidine (50 µg ml⁻¹) and tryptophan (50 µg ml⁻¹) were supplemented for growth of B. subtilis 168, B. subtilis DB430 and their derivatives.

For selection of plasmid-encoded or genome-integrated resistances, antibiotics were used at the following concentrations: ampicillin, 100 µg ml⁻¹ for E. coli; chloramphenicol, 5 µg ml⁻¹ for B. subtilis and 50 µg ml⁻¹ for E. coli; tetracycline, 8 µg ml⁻¹ for B. subtilis and 25 µg ml⁻¹ for E. coli.

LB-β-galactosidase indicator plates to identify LacZ-positive B. subtilis strains contained 0.03% (w/v) 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal).

2.2 Transformation

Competent cells of B. subtilis were transformed as described previously [16].

2.3 Construction of lipA and lipB deletion mutants

B. subtilis strains TEB1010 (ΔlipA), TEB1020 (ΔlipB) and TEB1030 (ΔlipAΔlipB) were constructed in a two-step procedure described by Hamilton et al. [9]. Deletion plasmids pO4ΔlipA and pO4ΔlipB (Table 1) were constructed by cloning DNA fragments located either 500 bp upstream and 500 bp downstream of lipA or lipB, respectively, into plasmid pORI240. Upstream (primer pairs mutA1/mutA2 and mutB1/mutB2) and downstream (primer pairs mutA3/mutA4 and mutB3/mutB4) regions were polymerase chain reaction (PCR)-amplified separately and afterwards fused in an overlap extension PCR reaction [10]. For this, the self-overlapping primers mutA2 (mutB2 for lipB) and mutA3 (mutB3) were designed which additionally contained stop codons in all six reading frames (Table 2). B. subtilis DB430 was transformed with either pO4ΔlipA or pO4ΔlipB and tetracycline resistant clones were selected. Transformants which contained the integrated plasmid were identified as LacZ-positive on X-Gal agar plates. Subsequently, transformants were grown in the absence of tetracycline to screen for LacZ-negative and tetracycline sensitive clones which were then checked for deletions of lipA and lipB by both PCR and Southern blotting.

2.4 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunodetection of LipA and LipB proteins

The supernatants from B. subtilis cultures grown in rich or minimal medium to the late exponential growth phase were isolated by centrifugation. The proteins were concentrated by TCA precipitation and separated by SDS–PAGE using a 5% stacking gel and a 15% separating gel. The proteins were Western blotted onto a PVDF (polyvinylidene difluoride) membrane and the lipolytic enzymes LipA and LipB were immunodetected using specific antibodies as previously described [6].

2.5 Enzymatic assays

Lipolytic activities were determined by using a spectrophotometric assay with p-nitrophenyl-caprylate as the substrate. The p-nitrophenyl-ester was dissolved in 10 ml isopropanol and mixed with 90 ml of S∅rensen phosphate buffer, pH 8 supplemented with sodium deoxycholic acid (207 mg) and gum arabic (100 mg). The final concentration of the substrate was 0.8 mM. For assaying secreted lipolytic activities of B. subtilis strains, about 10 to 100 µl culture supernatant was added to 2.5 ml of the substrate emulsion and after 30 min incubation at 37°C the absorbance at 410 nm was recorded with a Pharmacia LKB Novaspec II Photometer (Pharmacia, Freiburg, Germany). The enzymatic activity was calculated using a molar absorption coefficient of 15 000 M⁻¹ cm⁻¹.

The β-galactosidase activities of the B. subtilis lacZ-fusion strains were detected as described by Miller [20] using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate. ONPG turnover was recorded at a wavelength of 420 nm using a Pharmacia LKB Novaspec II Photometer (Pharmacia, Freiburg, Germany).

3 Results and discussion

The B. subtilis deletion mutants lipA (strain TEB1010) and lipB (strain TEB1020) as well as the wild-type (strain DB430) and the lipase-negative lipA-lipB double mutant (strain TEB1030) were cultured in rich and in minimal medium. Lipase activities in culture supernatants were determined spectrophotometrically with p-nitrophenyl caprylate as the substrate. At the end of the exponential growth phase the wild-type strain produced at least 20 U l⁻¹ of lipase in both media whereas the activity of the lipase-negative control strain (lipA-lipB double mutant) was 0,5–2 U l⁻¹, presumably caused by other hydrolytic enzymes (Fig. 1). Surprisingly, the lipB mutant strain B. subtilis TEB1020 which should still produce LipA also showed only background lipolytic activity in rich medium. In contrast, however, the lipA mutant TEB1010 did show the same activity as the wild-type strain. Both lipase proteins were detected in the wild-type strain by Western blotting using culture supernatants of strains grown in rich medium (Fig. 1B). These results demonstrate that LipB was the only enzymatically active extracellular lipase produced by B. subtilis cultured in LB medium supplemented with glucose whereas LipA was expressed and also secreted but remained enzymatically inactive. When the bacteria were grown in minimal medium, B. subtilis strain TEB1010 (ΔlipA) did not produce LipB as indicated by determination of enzyme activity (Fig. 1A) and by Western blotting (Fig. 1B). In contrast, enzymatically active LipA was produced and secreted.

In an attempt to clarify the striking difference in lipase production by B. subtilis grown in glucose-containing rich and minimal media we determined the pH values during growth. The LB medium showed a marked decrease in pH over the first 4 h of growth (Fig. 2A) with the initial pH value of about 7.0 rapidly decreasing to pH 5.0–5.5, probably resulting from an increased production of acidic glycolysis products such as acetate [24]. The pH value in minimal medium remained constant at the initial value of pH 7 because this medium contains potassium phosphate buffer (125 mM K₂HPO₄/KH₂PO₄ buffer, pH 7). The mechanism of LipA inactivation at low pH was further investigated by growing B. subtilis TEB1020 producing LipA as the only extracellular lipase in glucose-containing rich and minimal media buffered to pH 5.0 and 7.0. At pH 5.0, LipA was secreted but remained inactive, whereas enzymatically active LipA was produced at pH 7.0 (Fig. 2B,C). For B. subtilis TEB1010 which produces only LipB, the pH of the culture medium had essentially no influence on enzymatic activity (Fig. 2B,C). Previously, we had observed that purified LipA as well as LipB were inactivated after incubation at low pH values, but both proteins could be reactivated by incubation for at least 1 h at pH 11 [5]. Such a reactivation proved to be impossible with LipA as present in the culture supernatant of B. subtilis TEB 1020 grown at pH 5.0. Although the existence of a specific inhibitor was postulated for a phospholipase associated to the B. subtilis membrane [13,15], we assume that the inactivation of LipA at low pH may occur primarily by misfolding during or immediately after secretion across the B. subtilis cell wall.

Transcriptional lipA- and lipB-lacZ-fusions were constructed using the pDG268 lacZ-fusion vector (Table 1). Highest β-galactosidase activities [20] were detected at the end of the exponential growth phase with maximum activities of 7,5±3 Miller Units (MU) in rich and 50±5 MU in minimal medium for lipA-lacZ and 21±2,7 MU in rich medium for lipB-lacZ. B. subtilis containing the lipB-lacZ-fusion did not produce any β-galactosidase activity when cultured in minimal medium indicating that no transcription of the lipB gene occurred under these growth conditions as already suggested by the results of the Western blotting experiments (Fig. 1B).

Stress conditions such as high ethanol or salt concentrations affect the production of most of the degradative enzymes secreted by B. subtilis[16]. We have tested the influence of these conditions on the expression of lipA and lipB but ethanol at concentrations up to 4% (v/v) as well as NaCl (at concentrations up to 0.3 M) had no influence on the expression level of the corresponding lipase-reporter gene fusions. Another general feature of degradative enzyme regulation in B. subtilis is known as the glucose-driven catabolite repression which is mediated by cis-acting elements, the so-called catabolite responsive elements (CRE-box) and the DNA-binding protein CcpA (catabolite control protein) [11,19]. Although we have identified in the lipA gene a CRE-box [21,25], gene expression of both lipA and lipB proved to be independent from the glucose concentration in the growth medium (data not shown).

The lipA-gene expression of B. subtilis was repressed by amino acids upon growth in minimal medium (Fig. 3). No transcriptional activity was detectable at any time of growth when the bacteria were grown in minimal medium at a casamino acids concentration exceeding 1 mg ml⁻¹. As outlined above, the lipB gene was not expressed when the bacteria were grown in minimal medium. However, a β-galactosidase activity of 19±3 MU was recorded after addition to the growth medium of casamino acids at a final concentration of 1 mg ml⁻¹.

At present, the physiological role of the lipases LipA and LipB for B. subtilis is unknown. Our results clearly show that the expression of these lipases is differentially regulated, presumably at the transcriptional level, by the external culture pH as well as by the availability of amino acids. Further investigation is needed to identify other factors which may also be involved in this regulatory process.

Acknowledgements

This work was supported by the European Commission in the framework of the program Biotechnology (project no. BIO4-CT98-0249). U.B. is a recipient of a scholarship from the European Graduate College 795 entitled ‘Regulatory Circuits in Cellular Systems: Fundamentals and Biotechnological Applications’ funded by the Deutsche Forschungsgemeinschaft (DFG).

References

© 2003 Federation of European Microbiological Societies

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