Development of a New Integration Site within the Bacillus subtilis

Chromosome and Construction of Compatible Expression Cassettes

B. Härtl et al. (2001)

J. Bacteriol. 183: 2696-2699

Outline

1. Introduction

2. Objectives of the Experiments

3. Experiments

3.1 Construction of the lacA delivery expression vector pAX01

3.2 Construction of a lacA::spec chromosomal insertion mutation

3.3 Construction of the IPTG-inducible expression vector pA-spac

Outline

3.4 Regulation of the Expression of β-Gal from the lacA locus by the integrative expression vectors pAX01 and pA-spac

3.5 Integration of hrcA gene at the lacA locus and Regulation of the Expression of an inserted gene at the amyE locus

4. Conclusions

5. Take – Home – Lesson

6. Discussion

Introduction

• several different plasmids as cloning vectors in B. subtilis

→ disadvantage: replication via single-stranded DNA intermediate

→ instability of these plasmids

• alternative method: usage of integrative plasmids

→ widely used system: nonreplicating delivery vectors

Introduction

• ectopic integration: insertion of a foreign gene at a non-homologous site

• simplest form of the system:

→ antibiotic resistance marker gene + MCS enclosed by two halves of the amyE gene (amyE-front & amyE-back)

→ tranformation into B. subtilis cells

→ recombination at homologous sites via double- crossover

Objectives

• Generation of an additional integration site at the lacA locus in Bacillus subtilis

• Development of the two different delivery expression vectors pAX01 and pA-spac

• Creation of the two recipient strains IHA01 and IHA02

• Verification which of the both vectors works superior in comparison to each other

• Demonstration of the communication between the two loci lacA and amyE

Experiments

Construction of the lacA delivery expression vector pAX01

Three different fragments were produced: 1. lacA-front and lacA-back (generated seperately)

→ template: chromosomal DNA of B.subtilis 2. xylose expression cassette fused to the transcription

terminator → templates: pX and phage DNA 3. Erythromycin resistance gene (erm) → template: pMUTIN4 Assembling of the fragments by ligation with the usage of

restriction enzymes → pAX01

Construction of the lacA delivery expression vector pAX01

• modular structure of the delivery expression vector

→ greatly improved versatility

→ ability to adjust it to all sorts of applications

Construction of a lacA::spec chromosomal insertion mutation

• Integration of pAX01 can occur via single- or double-crossover event

→ single-crossover: integration of the whole vector at the lacA locus

→ double-crossover: only integration of the desired gene

→ Ermr - transformants

Construction of a lacA::spec chromosomal insertion mutation

• Discrimination of these transformants:

→ creation of a recipient strain that contains a Specr marker in the chromosomal DNA at the lacA locus

IHA01 (lacA::spec)

• previously described recipient strain for the amyE locus: AM01 (amyE::cat)

Construction of a lacA::spec chromosomal insertion mutation

• Combination of lacA::spec and amyE::cat in a single strain:

→ preparation of chromosomal DNA from IHA01 and transformation into AM01

→ IHA02

→ allows insertion of two different DNA sequences at

both loci within the same strain

Construction of the IPTG-inducible expression vector pA-spac

• E. coli lac promoter system as the most widely used expression system in B. subtilis

→ negative control by lac-repressor

→ IPTG as an inducer (inactivates repressor)

• Construction of the pA-spac vector:

→ creation of a lac expression cassette (includes lacI gene and lac promoter-operator region)

→ insertion into pAX01 → pA-spac

Construction of the IPTG-inducible expression vector pA-spac

Verification which of the both vectors (pAX01 and pA-spac) works superior in comparison to each other

• There are two expression cassettes that would be compatible for a new integration site within the B. subtilis chromosome

→ pAX01 and pA-spac

Verification which of the both vectors (pAX01 and pA-spac) works superior in comparison to each other

• Question: Which of both vectors works superior?

1. insertion of a reporter gene (bgaB; encodes for heatstable β-Gal) into both vectors

→ pAX01-bgaB and pA-spac-bgaB

2. transformation into IHA01

→ IHA01-Xyl-bgaB and IHA01-Spac-bgaB

3. measurement of the activity of the enzyme in absence and in presence of an inducer

Regulation of the Expression of β-Gal using the vector pA-spac

Regulation of the Expression of β-Gal using the vector pA-spac

• presence of IPTG:

→ no influence on the growth if the strain

→ steady increase of the activity of β-Gal over time

→ 0,1 mM IPTG was sufficient to reach full induction rate

• absence of IPTG:

→ slow increase of the activity

→ there is some leakiness in the system

Regulation of the Expression of β-Gal using the vector pAX01

Regulation of the Expression of β-Gal using the vector pAX01

• presence of Xylose:

→ no influence on the growth if the strain

→ high increase of the activity of β-Gal

→ decline after 200 min (consumption of the sugar)

→ 0,5 % Xylose was sufficient to reach full induction rate

• absence of Xylose:

→ no activity

Verification which of the both vectors (pAX01 and pA-spac) works superior in comparison to each other

The Xylose – inducible system is superior to the

IPTG – inducible system because of two reasons:

1. There is no expression of the bgaB gene in the absence of the inducer xylose

2. It results in a higher inducction rate

(40 – folds versus 15 – folds)

Communication between the two loci lacA and amyE

• amyE locus existed already as an integration site

• lacA locus was developed as a new integration site

• Question:

Is this new integration site useful in obtaining regulated expression of a gene that was integrated at the amyE locus in the same strain?

Communication between the two loci lacA and amyE

• creation of a strain that contains at the lacA locus a gene for a repressor affecting the operator of the gene that was integrated at the amyE locus

→ generation of the hrcA repressor gene and ligation into pAX01 → pAX01-hrcA

→ transformation into AM01 and homologous recombination (AM01: no own hrcA gene + reporter gene bgaB at the amyE locus ) → IHA13

Communication between the two loci lacA and amyE

Communication between the two loci lacA and amyE

• Measurement of the β-Gal activity

• Absence of Xylose:

→ no expression of the hrcA repressor gene

→ high expression of the bgaB gene

→ increase of the β-Gal activity

• Presence of 0,5 % Xylose:

→ decay of the activity to about 10 U

→ about 3 h after the addition of the inducer the β- Gal activity began to rise

Communication between the two loci lacA and amyE

Result:

Regulated expression from one chromosomal locus can influence the transcription of a distant one.

Conclusions

Conclusions

• lacA locus as a new integration site for ectopic integration

→ codes for very weakly expressed β-Gal • lacA::spec as a chromosomal insertion mutation in the

IHA01 strain (for easy screening) • creation of two delivery expression vectors:

pAX01 & pA-spac • measuring the activity of the β-Gal results in the fact

that pAX01 is the better vector for regulative expression

• the expression of the hrcA gene at the lacA locus was able to affect the expression of the bgaB reporter gene at the amyE locus

Take – Home - Message

The lacA locus was developed as a new site fo ectopic integration within the B. subtilis chromosome and the integration of a regulatory gene at this site is able to

control the expression of a transcriptional fusion at the amyE locus.

Questions

1. Auf welchem Prinzip beruht die Integration eines gewünschten Gens in ein chromosomales?

2. Was versteht man unter „ektopischer Integration“?