Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 ....

Results

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-.......

Discussion

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.......

Materials

List of materials used in this article

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