Genetic Manipulation of a Naturally Competent Bacterium, Helicobacter pylori

Abstract

Genetic manipulation of Helicobacter pylori facilitates characterization and functional analysis of individual H. pylori genes. This chapter discusses the methods involved in H. pylori chromosomal DNA isolation, mutagenesis of individual genes, and natural transformation.

1. Introduction

Helicobacter pylori is a Gram-negative bacterial pathogen that inhabits the human stomach. Infection can lead to a number of diverse disease outcomes, including gastric and duodenal ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. It is well documented that both H. pylori virulence determinants as well as host factors heighten the risk for H. pylori-associated disease. H. pylori exhibits remarkable genetic diversity as a species, as evidenced by variation of gene order and genetic content of strains, mosaic nature of genes, and sequence diversity within conserved genes (1–4). H. pylori has a panmictic population structure, indicative of frequent genetic exchange among strains. Additionally, there is evidence that recombination between H. pylori strains can occur in vivo during naturally occurring mixed infections. Analyses of complete genomic sequences from a number of H. pylori isolates indicate that this organism has incorporated genetic material from other organisms during the course of its existence. This exchange of genetic material (e.g., horizontal gene exchange) can occur via three classical mechanisms: conjugation, transduction, and transformation. Conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact, while transduction involves the transfer of genetic material between bacteria via bacteriophage. Transformation is the genetic alteration of a cell resulting from the uptake, incorporation, and expression of exogenous genetic material. H. pylori is, in general, naturally competent, whereby transformation is a relatively frequent occurrence. Approximately 75% of all H. pylori strains are naturally competent for uptake of H. pylori chromosomal DNA, and this feature allows investigators to easily genetically manipulate this pathogen. Characterization of experimentally generated mutants forms the basis of modern microbial genetics and is particularly useful for determining the function of individual genes by comparing the phenotype of a mutant in which the gene is no longer expressed to the wild-type strain. Targeted or random mutagenesis of H. pylori involves a technique termed allelic exchange mutagenesis, in which cloned fragments of H. pylori DNA are inactivated in Escherichia coli and then introduced into H. pylori for allelic exchange by natural transformation. This chapter discusses the specific materials and methods required for creating genetic mutations in the bacterial pathogen, Helicobacter pylori.

2. Materials

2.1. H. pylori Chromosomal DNA Isolation

1. Culture of Helicobacter pylori (plate or broth culture).

2. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0 (store at 4°C).

3. 10% Sodium dodecyl sulfate (SDS) (store at room temperature).

4. 20 mg/ml Proteinase K (store aliquots at –20°C).

5. 5 M NaCl (store at room temperature).

6. CTAB/NaCl solution (store at room temperature):

   1. Dissolve 4.1 g NaCl in 80 ml of dH₂O.
   2. Add 10 g CTAB slowly while heating and stirring.
   3. Adjust final volume to 100 ml.

7. 24:1 Chloroform/isoamyl alcohol (store at 4°C).

8. 25:24:1 Phenol/chloroform/isoamyl alcohol (store at 4°C).

9. Isopropanol (store at 4°C).

10. 70% Ethanol (store at 4°C).

2.2. H. pylori Mutagenesis

1. H. pylori chromosomal DNA.

2. PCR reagents: 10× PCR amplification buffer with MgCl₂, 20 mM dNTP mixture, 5 μM primers, Taq DNA polymerase, and molecular biology grade H₂O.

3. Commercially available PCR purification kit or, alternatively, reagents for standard method of DNA purification and isolation.

4. PCR cloning vector.

5. Ligation reagents: T4 DNA ligase, ligation buffer, and molecular biology grade H₂O.

6. Restriction enzyme reagents: Restriction enzyme(s), 10× restriction enzyme buffer(s), and molecular biology grade H₂O.

7. Competent E. coli cells, Luria broth (LB), and antibiotic stock solutions (see Note 1).

8. Commercially available plasmid preparation kit or, alternatively, reagents for standard alkaline lysis method.

9. Agarose, ethidium bromide, and gel electrophoresis equipment.

2.3. Natural Transformation

1. Brucella broth (BB) and Brucella agar (BA):

   1. Brucella broth: BB + 10% fetal calf serum (FCS).
   2. Brucella agar plates: BA + 10% FCS.

2. Antibiotic stock solutions (see Note 2).

3. Tryptic soy agar with 5% sheep blood (TSA blood) plates (commercially available).

4. Sterile 1× PBS.

5. Naturally competent strain of Helicobacter pylori.

3. Methods

3.1. H. pylori Chromosomal DNA Isolation (5)

1. Grow H. pylori on TSA blood plates overnight (~16–18 h) at 37°C with 5% CO₂.

2. Suspend H. pylori cells from one plate in 900 μl of TE buffer.

3. Pellet bacteria by centrifugation at 8,000 rpm for 5 min.

4. Discard the supernatant and resuspend the bacterial pellet in 567 μl of TE buffer.

5. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K to yield a final concentration of 100 μl/ml proteinase K in 0.5% SDS. Mix thoroughly and incubate for 1 h at 37°C.

6. Add 100 μl of 5 M NaCl and mix thoroughly.

7. Add 80 μl of CTAB/NaCl solution. Mix thoroughly and incubate for 10 min at 65°C.

8. Add an equal volume of 24:1 chloroform/isoamyl alcohol (~700–800 μl), mix thoroughly, and subject to centrifugation at 8,000 rpm for 5 min.

9. Transfer aqueous phase (supernatant) to a new microcentrifuge tube, leaving the interface behind.

10. Add an equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol to the aqueous phase, mix thoroughly, and subject to centrifugation at 8,000 rpm for 5 min.

11. Transfer aqueous phase (supernatant) to a new microcentrifuge tube and add 0.6 volume isopropanol to precipitate the nucleic acids. Shake gently until stringy, white DNA precipitate appears.

12. Pellet the DNA by centrifugation at 8,000 rpm for 5 min and then remove isopropanol.

13. Wash the DNA with 100 μl 70% ethanol to remove residual CTAB.

14. Pellet the DNA by centrifugation at 8,000 rpm for 5 min and then remove and discard ethanol.

15. Dry DNA pellet in lyophilizer or, alternatively, open tube and leave on bench or in hood.

16. Once DNA pellet is dry, dissolve DNA in 100 μl TE buffer.

3.2. H. pylori Mutagenesis

3.2.1. Site-Specific Mutagenesis by Overlap Extension (6, 7)

1. Design and synthesize oligonucleotide primers, Forward (F), Reverse Mutagenic (RM), Forward Mutagenic (FM), and Reverse (R), based on the known gene sequence within the H. pylori strain of interest (Fig. 1) (see Note 3).

2. In a sterile PCR tube, set up PCR1 by mixing the following reagents:

   H. pylori chromosomal DNA	~100 ng
   10× amplification buffer	10 μl
   20 mM dNTP mixture	1 μl
   5 μM F primer (30 pmol)	6 μl
   5 μM RM primer (30 pmol)	6 μl
   Taq DNA polymerase	1–2 units
   Molecular biology grade H2O	To 100 μl

3. In another sterile PCR tube, set up PCR2 by mixing the following reagents:

   H. pylori chromosomal DNA	~100 ng
   10× amplification buffer	10 μl
   20 mM dNTP mixture	1 μl
   5 μM FM primer (30 pmol)	6 μl
   5 μM R primer (30 pmol)	6 μl
   Taq DNA polymerase	1–2 units
   Molecular biology grade H2O	To 100 μl (see Note 4)

4. Amplify the nucleic acids in PCR1 and PCR2 by using the denaturation, annealing, and extension conditions listed below:

   1. Initial Denaturation: 5 min at 95°C.
   2. 30–35 repeated cycles:

      1. Denaturation: 1 min at 95°C.
      2. Annealing: 1 min at determined annealing temperature. NOTE: The annealing temperature should be approximately 5–10°C below the melting temperature (Tm) of primers.
      3. Extension: 1 min at 72°C (1 min/kb).
   3. Final Extension: 10 min at 72°C.

5. Analyze 5% of each of PCR1 and PCR2 on a 1% ethidium bromide agarose gel and estimate the concentration of amplified product from PCR1 and PCR2.

6. Purify the PCR1 and PCR2 products using a commercially available PCR purification kit.

7. In a sterile PCR tube, set up PCR3 by mixing the following reagents:

   Amplification product from PCR1	~50 ng
   Amplification product from PCR2	~50 ng
   10× amplification buffer	10 μl
   20 mM dNTP mixture	1 μl
   5 μM F primer (30 pmol)	6 μl
   5 μM RM primer (30 pmol)	6 μl
   Taq DNA polymerase	1–2 units
   Molecular biology grade H2O	To 100 μl

8. Amplify the nucleic acids using the denaturation, annealing, and extension conditions listed in step 4.

9. Analyze 5% of the PCR on a 1% ethidium bromide agarose gel, estimate the concentration of amplified product from PCR3, and then purify the PCR3 product using commercially available PCR purification kit (see Note 5).

10. Clone PCR3 product into PCR cloning vector and transform competent E. coli cells. NOTE: Blue/white screening is a useful tool to identify positive E. coli transformants/clones.

11. Grow E. coli cultures overnight at 37°C with shaking at ~100 rpm and then harvest plasmid using commercially available plasmid preparation kit.

12. Digest plasmid with appropriate restriction enzyme to open plasmid at engineered, novel restriction site within cloned PCR3 product.

13. Ligate antibiotic resistance-encoding cassette within H. pylori gene of interest.

14. Transform competent E. coli cells with ligated vector.

15. Grow overnight at 37°C with shaking at ~100 rpm and then select for antibiotic-resistant clones by growing E. coli on selective antibiotic LB plates.

16. Harvest E. coli and isolate antibiotic-resistant plasmids for Helicobacter pylori transformation.

17. To confirm directional cloning, use restriction enzymes to digest PCR cloning vector at various sites within the multiple cloning site.

    1. Digest with enzymes to excise PCR3 product.
    2. Digest with enzymes to excise antibiotic resistance-encoding cassette.
    3. Digest with multiple enzymes to determine directionality of the PCR3 product and antibiotic resistance-encoding cassette.

18. Analyze products of restriction digestions on a 1% ethidium bromide agarose gel and proceed if restriction maps are correct (see Note 6).

Fig. 1.

Schematic representation of site-specific mutagenesis by overlap extension (7). Forward (F) and Reverse Mutagenic (RM) primers are used to amplify the upstream region of a specific target gene in PCR1. Forward Mutagenic (FM) and Reverse (R) primers are used to amplify the downstream region of a specific target gene in PCR2. Mutagenic primers are designed to encode a novel restriction site, as shown. Following the primary PCR steps, products from PCR1 and PCR2 are used in conjunction with Forward (F) and Reverse (R) primers in PCR3, where the overlapping sequences serve to prime this reaction. The product of PCR3 is then cloned into a PCR cloning vector and subsequently digested at the novel restriction sites. An antibiotic resistance-encoding cassette (as shown) is then inserted into this site and the construct is then used for H. pylori transformation.

3.3. Natural Transformation

3.3.1. Plate Transformation

1. Collect H. pylori cells from an overnight (~16–18 h) TSA blood plate in 1 ml of sterile 1× PBS.

2. Harvest bacteria by centrifugation at 8,000 rpm for 5 min.

3. Resuspend bacteria in 100 μl of Brucella broth.

4. Spot 25 μl of H. pylori cells onto a TSA blood plate.

5. Add ~1 μg of antibiotic-resistant plasmid preparation to H. pylori.

6. Incubate TSA plate right-side-up overnight at 37°C with 5% CO₂.

7. Following overnight incubation, use sterile swab to transfer H. pylori spot to Brucella agar plates with antibiotic for selection of positive transformants.

8. Incubate for ~5 days or until colonies appear.

9. Expand colonies on Brucella agar selective antibiotic plates and freeze H. pylori-positive transformants at –80°C for future use.

10. Confirm H. pylori mutations by PCR amplification of chromosomal DNA and sequence analysis of the gene of interest.

3.3.2. Broth Transformation

1. Inoculate Brucella broth with H. pylori (either from a 24-h TSA blood plate or from an H. pylori starter overnight culture). Cultures should be started at an OD₆₀₀ of ~0.1–0.2 (see Note 7).

2. Add ~1–2 μg of antibiotic-resistant plasmid DNA and grow overnight (~16–18 h) at 37°C with 5% CO₂.

3. Measure OD₆₀₀ (see Note 8).

4. Remove 1 ml of H. pylori culture and harvest by centrifugation at 8,000 rpm for 5 min.

5. Resuspend bacteria in 100 μl of Brucella broth.

6. Plate 100 μl on Brucella agar antibiotic plates and incubate right-side-up overnight at 37°C with 5% CO₂.

7. Turn plate upside down and continue to incubate at 37°C with 5% CO₂ for ~5 days or until colonies appear.

8. Expand colonies on Brucella agar selective antibiotic plates and freeze positive transformants at –80°C for future use.

9. Confirm H. pylori mutations by PCR amplification of chromosomal DNA and sequence analysis.