Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Podsumowanie

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Streszczenie

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir⁺ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir⁺ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Wprowadzenie

Prototrophic bacteria grow in M-9 minimal salts medium containing glucose (M-9 medium), converting glucose through central carbon metabolism pathways to generate precursors, such as amino acids, nucleic acids and vitamins, for biosynthesis¹. M-9 medium contains ammonium chloride as a nitrogen source, sodium and potassium phosphate as a buffer and phosphorous source, magnesium sulfate as a sulfur source and glucose as a carbon and energy source. Luria-Bertani (LB) medium is rich in amino acids from tryptone and in vitamins and growth factors from yeast extract. It supports the growth of auxotrophs that cannot synthesize amino acids, vitamins and other growth factors required for growth on M-9 medium. Thus, prototrophs will grow in LB and M-9 medium, whereas auxotrophs will grow in LB medium but not in M-9 medium. By introducing mutations into a prototrophic population of bacteria and identifying mutated genes that cause auxotrophic phenotypes, it is possible to obtain a better understanding of the metabolism in a bacterial strain.

Transposon mutagenesis can be used to identify many of the genes required for growth on glucose in M-9 medium. Transposons insert themselves randomly into the host genome². By spotting transposon transformants on LB agar plates and replica plating them onto M-9 medium agar plates, it is possible to screen for auxotrophs. Interrupted genes are identified through gene rescue. This study uses a commercially available Tn5-derived transposome which is shipped as a solution of a linear transposon DNA segment mixed with transposase protein. The DNA segment lacks a transposase gene but contains a gene for kanamycin resistance, an R6Kγ replication origin and two mosaic motifs which are DNA sequences for transposase binding at each end of the segment^3,4. Since the transposase protein is added directly to the DNA, the DNA segment alone is defined as the transposon, and the DNA/transposase protein complex is defined as the transposome. The transposome is transformed by electroporation⁵ into a kanamycin sensitive host (Figure 1A). Colonies that grow on LB agar plates containing kanamycin (LB-kan) have transposon inserts (Figure 1B), and replica plated transformants that fail to grow on M-9 medium agar plates containing kanamycin (M-9-kan) are auxotrophs (Figire 1C). Genomic DNA from a mutant is purified and partially digested with the 4-base cutting restriction endonuclease, BfuC I (Figure 1D). The ligated DNA is transformed into a strain of Escherichia coli (E. coli) that contains the pir gene (Figure 1E). This gene allows the new plasmid containing the transposon and flanking host chromosomal region to replicate in E. coli⁶. The kanamycin resistance gene serves as a selectable marker for the new plasmid. Finally, sequencing using primers complementary to each end of the transposon and Basic Local Alignment Search Tool (BLAST) analysis^7,8 of the resulting sequence are used to determine the identity of the interrupted genes.

This transposome mutagenesis strategy provides three advantages³. First, since the transposase protein is bound directly to the transposon, insertion does not depend on expression of the transposase gene within the host. Once the transposon incorporates itself into the host genome, the transposase is degraded, preventing additional movement of the transposon. However, additional movement cannot be prevented if the host possesses an endogenous Tn5 transpositional element. Second, introduction of the transposome by electroporation makes it possible to use it in a wide variety of hosts. It also eliminates the need to introduce the transposon by bacterial conjugation or by viral infection. Both processes require host susceptibility. Third, inclusion of the kanamycin resistance gene and the R6Kγ replication origin in the transposome makes it easier to identify the interrupted gene. Transposon-interrupted regions can be stored and sequenced as plasmids, eliminating the need to use the inverse polymerase chain reaction (PCR) technique for gene identification.

The protocol presented in this video describes each step for transposon mutagenesis of Enterobacter sp. YSU⁹ using a transposome from its introduction into the bacterial cells to the identification of the putative gene it interrupted. In addition to previously published protocols^3,4,10, detailed methods for using replica plating to screen for auxotrophs are presented. This mutagenesis technique may be used for investigating other phenotypes, such as antibiotic and metal resistances, in different types of bacteria, for identifying the minimal number of genes required for growth under defined culture conditions in synthetic biology studies, or for teaching a laboratory component of a genetics or microbial physiology course.  

Protokół

1. Electroporation of Competent Cells^5,11

1. Dilute an O/N LB culture of Enterobacter sp. YSU 1/20 into 50 ml of fresh LB medium and grow with shaking at 120 rpm and 30 °C to an O.D. (600 nm) between 0.4 and 0.6. Optionally, grow other bacterial strains at their optimal growth temperature.
2. Chill the cells on ice for 5 min and centrifuge at 4 °C and 7,000 x g for 5 min.
3. Discard the supernatant, resuspend the cells in 50 ml of sterile ice cold water and centrifuge at 4 °C and 7,000 x g for 5 min. Repeat this step.
4. Discard the supernatant and resuspend the cells in a volume of ice cold water equal to the cell pellet volume. For long term storage, wash the cells with 10 ml of ice cold 10% (v/v) glycerol, resuspend in an equal pellet volume of ice cold 10% (v/v) glycerol, store at -80 °C, and thaw on ice before electroporation.
5. Add 0.5 µl of the transposome to 40 µl of cells. The commercially available transposome solution is supplied at a DNA concentration of 0.33 µg/µl. Although 0.33 µg is recommended, 0.165 µg DNA is sufficient.
6. Place the cell/transposome mixture in an ice cold, 0.2 mm, electroporation cuvette and tap the mixture to the bottom of the cuvette. Shock the cells at 25 µF, 200 Ω, and 2.5 kV. To ensure that the cells remain chilled, store the electroporation cuvettes in a -20°C freezer before use.
7. Immediately, add 960 µl of filter sterilized super optimal broth with catabolite repression (SOC) medium. Mix by pipetting up and down and transfer the cells to a sterile 1.5 ml microfuge tube.
   NOTE: The cells begin to die after the shock, but the salt in the SOC medium allows them to recover. To economize, it is not necessary to add transposome or to shock the negative control cells. Just add 960 µl of sterile SOC medium to it.
8. Incubate the cells at 30 °C for 45-60 min with shaking at 120 rpm. This provides time for the transposome to recombine with the host genome and for the cells to express kanamycin resistance.
9. Spread 100 µl of cells on a LB-kan agar plate and incubate the plate O/N at 30 °C. Store the remaining transformation mix in the refrigerator at 4 °C. Spread additional amounts at a later date up to 2 weeks after the initial electroporation if required.

2. Gridding of Transformants

1. Tape a grid to the lid of an empty 100 x 15 mm petri dish. Copy a grid from “A Short Course in Bacterial Genetics”¹².
2. Draw a line on the bottom of an LB-kan agar plate. Use a small piece of tape to anchor it to the gridded lid so that the grid is visible through the agar. Ensure that the line on the bottom of the plate is aligned with the top, center of the grid. Anchoring the plate with tape keeps it from sliding, allowing for even gridding. The line facilitates colony identification after replica plating.
3. Pick a single transformant with a sterile toothpick and spot it in the center of a square in the grid. Just touch the colony and the spot. Do not dig the toothpick into the agar. To avoid contaminating the plate, handle the toothpick only at one end.
4. Spot another transformant in an adjacent square as in step 2.3. When the plate is full, incubate it O/N at 30 °C. This is the master plate.

3. Replica Plating to Determine the Auxotroph Phenotype¹²

1. For each plate that was gridded, make a stack of three fresh agar plates numbered one through three: one for LB-kan, two for M-9-kan and three for LB-kan. Dry the plates upside down, O/N, at 37 °C before use.
2. Draw a line on the top of each plate as in step 2.2.
3. Wipe the replica plating tool with 70% ethanol, place a sterile velveteen square on top of the replica plating tool and clamp the velveteen square down. Hands are teeming with microbes even after washing them. Prevent the introduction of contaminating microbes by handling the velveteen square by its edge.
4. Align the line on the master plate with a line drawn on the clamp and place the plate on top of the velveteen square. Apply gentle pressure to transfer the bacteria from the plate to the velveteen square. Be careful not to smear the bacteria. Remove the master plate from the velveteen square.
5. Align the line drawn on plate number one with the line on the clamp and place it on top of the velveteen square. Apply gentle pressure to transfer the bacteria from the velveteen square to the plate. Remove plate number one.
6. Discard the velveteen square into a beaker of water and place a clean, sterile velveteen square on the replica plating tool as in step 3.3. This step prevents over-inoculation which causes breakthrough growth and false positive results.
7. Align the line on plate number one with the line on the clamp and place it on top of the new velveteen square. Apply gentle pressure to transfer the bacteria from plate number one to the velveteen square. Remove plate number one.
8. Align the line drawn on plate number two with the line on the clamp and place it on top of the velveteen square. Apply gentle pressure to transfer the bacteria from the velveteen square to plate number two. Remove plate number two.
9. Repeat step 3.8 for plate number three. The third plate is a control for complete transfer from the master plate to the other two plates. Discard the velveteen square into a beaker of water. To reuse the velveteen squares, autoclave the beaker containing the contaminated velveteen squares, wash them, dry them, wrap them in aluminum foil and re-autoclave them.
10. Incubate the plates at 30 °C O/N. Colonies that grow on both LB-kan agar plates but fail to grow on M-9-kan plates are considered to be auxotrophs (Figure 2).
11. Streak out auxotrophs from plate number one onto a fresh LB-kan agar plate, and incubate the plate at 30 °C O/N.
12. Streak out for isolation 3 colonies from the streak plate in step 3.11 onto a fresh LB-kan plate. Incubate the plate at 30 °C O/N. This ensures that the mutant is well isolated. Divide the plate into thirds and streak out each colony in one section.
13. Spot colonies from the streaked out colonies derived from step 3.12 onto a fresh LB-kan plate to form a grid as in steps 2.1-2.4. Each mutant is being re-tested in triplicate.
14. Replicate plate again as in steps 3.1-3.10 to confirm the auxotroph phenotype.

4. Gene Rescue

1. Grow a 3 ml O/N culture of an isolated mutant colony from step 3.13 in liquid LB-kan medium at 30 °C. Purify genomic DNA from 1 ml of culture using a commercially available genomic DNA purification kit.
2. Set up a mixture of 0.025 U BfuC I restriction endonuclease and 14 µl of 1 μg/μl genomic DNA in a 20 µl reaction on ice. Since BfuC I cuts the transposon at twelve different sites, it may be more efficient to use restriction endonuclease, Bfa I or Hae III, which cuts the transposon at two and three different sites, respectively.
3. Incubate the reaction at 37 °C for 25 min and then at 80 °C for 20 min to inactivate the enzyme. Analyze 3 µl of the partially digested DNA on a 0.8% agarose gel (Figure 3). A successful partial digestion results in a smear from above 10 kb down to the bottom of the gel.
4. Ligate 15 µl of partially digested genomic DNA using 800 units of T4 DNA ligase in a 500 µl reaction. Incubate the reaction O/N at 4 °C. The large reaction volume maximizes the ligation of single fragments to themselves and minimizes interactions between two or more DNA fragments.
5. Precipitate the ligated DNA by adding 50 µl of 3 M sodium acetate, pH 5.5, and 1 ml of 95% ethanol and incubating it at -20°C for 20 min.
6. Microfuge the DNA at 4 °C and 13,500 x g for 10 min, wash the pellet with 70% ethanol, dry it in a centrifugal vacuum concentrator, and resuspend it in 10 µl of nuclease free water. Alternatively, the DNA may be air dried at RT for 15 min.
7. Use 4 µl of resuspended DNA to transform E. coli strain ECD100D pir or ECD100D pir116 by electroporation as in section 1. The R6kγ replication origin requires a bacterial strain with a pir gene to replicate⁶. The pir strain results in low copy plasmids, and the pir116 strain contains a pir mutant gene that results in high copy plasmids. Each transformant contains a new plasmid with the transposon and a region of the interrupted chromosome (Figure 1E).
8. Inoculate 5 ml of LB-kan medium with a single colony, grow it O/N with shaking at 120 rpm and 37 °C, and purify plasmid DNA from the entire O/N culture using a commercial kit.
9. Digest 14 µl of the plasmid using the restriction endonuclease Xho I. Make sure the final volume of the reaction is 20 µl. This enzyme recognizes a site in the middle of the transposon at the 5’ end of the kanamycin resistance gene. Analyze 10 µl of the undigested and digested plasmid on a 0.8% agarose gel (Figure 3). The plasmid consists of the 2 kb transposon plus flanking host DNA.

5. DNA Sequencing

1. Sequence the plasmid using a commercially available sequencing kit, primers that are homologous to each end of the transposon, and a capillary sequencing analysis system. Alternatively, the plasmid may be shipped to an outside institution for sequencing.
2. Use the molecular weight standard (1 kb ladder) in the gel to estimate the size of the plasmid. Then, estimate the number of nanograms (ng) of plasmid that is required for 50 fmol.
3. Measure the concentration of the plasmid DNA using a spectrophotometer at wavelengths of 260 nm (A₂₆₀) and 280 nm (A₂₈₀). Ensure the light path length through the cuvette is 1 cm. Determine the concentration by multiplying the absorbance at 260 nm by 50 ng/µl. High quality plasmid DNA will have an A₂₆₀/ A₂₈₀ ratio between 1.8 and 2.0.
4. The volume of DNA required for each sequencing reaction is the number of ng required for 50 fmol divided by the concentration of the plasmid. Mix the volume of required plasmid with water to bring the total volume up to 10 µl.
5. Heat the plasmid DNA/water mixture at 96 °C for 1 min and then cool it to RT.
6. Add 8 µl of master mix from the sequencing kit and 2 µl of 1.6 μM of one of the sequencing primers.
7. Follow the protocol from the sequencing kit for the thermal cycler program and reaction cleanup.
8. Analyze each sample using a capillary DNA analysis system.
9. Use a commercially available software package to view the DNA sequence. Search for the sequence, “5’-GAGACAG-3’,” which is the last 7 bp of the transposon (Figure 4). The sequence before the 5’ end of this 7 bp segment belongs to the transposon. The sequence after the 3’ end of this 7 bp segment belongs to the interrupted gene.
10. Determine the possible function of the interrupted gene using the Basic Local Alignment Search Tool (BLAST) ^7,8. Use the following link: http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch. Paste the sequence of the interrupted gene into the query box, select “others” under the database and click on “BLAST” at the bottom of the page. When the result appears, scroll down the page to view the alignment results (Figure 5).

Wyniki

Transformation of Enterobacter sp. YSU by electroporation with the transposome initiated random genome insertion into the host genome (Figure 1A,B). A successful electroporation yielded several thousand transformants which grew on LB-kan plates. To obtain 300-400, well-spaced colonies per plate, the amount of transformation mixture spread on each LB-kan agar plate was optimized. Each transformant contained at least one transposon insert, but it was not clear if an important gene for growth on M-...

Dyskusje

Transposon mutagenesis using a transposome is an efficient tool for generating auxotrophs in Enterobacter sp. YSU and other types of Gram negative and Gram positive bacteria^3,4. The process was initiated by introducing the Tn5-derived transposome into the host by electroporation. To identify colonies with inserts, the untransformed target strain had to be sensitive to kanamycin in order to select for the resistance marker carried by the transposon. Some hosts produce restriction endonucleases...

Materiały

Name	Company	Catalog Number	Comments
EZ-Tn5 R6Kγori/KAN-2 Tnp Transposome Kit	Epicentre (Illumina)	TSM08KR
EC100D pir+ Electrocompetent E. coli	Epicentre (Illumina)	ECP09500	Capable of replicating plasmids with an R6Kγ replication origin at a low copy number
EC100D pir-116 Electrocompetent E. coli	Epicentre (Illumina)	EC6P095H	Capable of replicating plasmids with an R6Kγ replication origin at a high copy number
5X M-9 Salts	Thermo Fisher	DF048517
Lennox LB Broth	Thermo Fisher	BP1427-2
Agar	Amresco, Inc.	J637-1KG	Solid media contained 1.6% (w/v) agar
Kanamycin Sulfate	Amresco, Inc.	0408-25G	When required, media contained 50 μg/ml kanamycin sulfate
D-Glucose Monohydrate	Amresco, Inc.	0643-1KG
Yeast Extract	Amresco, Inc.	J850-500G
Tryptone	Amresco, Inc.	J859-500G
KCl	Sigma-Aldrich	P4504-500G
NaCl	Amresco, Inc.	X190-1KG
MgCl2	Fisher Scientific	BP214-500
MgSO2	Fisher Scientific	BP213-1
Super Optimal Broth with Catabolite Repression (SOC) medium			0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM MgSO4 and 20 mM Glucose
BfuC I	NEB	R0636S	Partial digestion of genomic DNA
Xho I	NEB	R0146S	Plasmid digestion
T4 DNA Ligase	NEB	M0202S
Nuclease Free Water	Amresco, Inc.	E476-1L	For dissolving precipitated DNA and restriction endonuclease reactions
GenomeLab DTCS - Quick Start Kit	Beckman Coulter	608120	DNA sequencing
Wizard Genomic DNA Purification Kit	Promega	A1120A
Wizard Plus SV Minipreps DNA Purification System	Promega	A1460	Plasmid purification
Replica Plating Block	Thermo Fisher	09-718-1
Velveteen Squares	Thermo Fisher	09-718-2	Replica plating
PetriStickers	Diversified Biotech	PSTK-1100	Grid for replica plating
Petri Dishes	Thermo Fisher	FB0875712
Gene Pulser II	BioRad		Electroporation
Electroporation Cuvettes – 2 mm	BioExpress	E-5010-2
CentriVap DNA Vacuum Concentrator	Labconco	7970010	Drying DNA
CEQ 2000XL DNA Analysis System	Beckman Coulter		DNA sequencing
Vector NTI Advance 11.5.0	Life Technologies	12605099	DNA sequence analysis