Genetic Footprinting in Bacteria

Abstract

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage transposon delivery system.Large-scale sequencing of the genomes of many different microorganisms has yielded information for thousands of genes. However, the functions of a significant number of these genes remain unknown. Conventional knockout techniques have been used to examine the effect of deleting or disrupting a gene, but these techniques are difficult to apply on a genomic scale. Genetic footprinting allows the rapid, simultaneous determination of the importance of a large number of genes required for growth under a chosen condition (19,20).Genetic footprinting is a three-step process involving transposon mutagenesis, outgrowth of the mutagenized cell population, and analysis of the fate of cells carrying mutations in specific genes. The first step involves insertional mutagenesis using a transposon that inserts randomly throughout the genome. Following this period of mutagenesis where transposase expression is induced, a sample of the initial population, designated T0 (time zero), is taken. The second step is growth of the mutagenized T0 culture over many population doublings under conditions that repress transposase expression and plasmid replication. The third step is the comparative evaluation of transposon insertions present within specific genes based on analysis of PCR products generated from DNA isolated from the T0 mutagenized culture versus samples from the outgrowth culture (e.g., T15, T30, and T45 [15, 30, 45 population doublings, respectively]). Bacteria containing transposon insertions in genes that are important for the fitness or viability of the organism under a specific growth condition will not be represented in the outgrowth population, and therefore loss or diminution of PCR products corresponding to insertions within that gene will be observed. By measuring the loss of gene-s...

From Genetic Footprinting to Antimicrobial Drug Targets: Examples in Cofactor Biosynthetic Pathways

“…This may be due to the fact that these genes can tolerate transposon inserts within certain restricted loci without a detrimental effect on the corresponding gene product. Similar phenomena have been reported in other genetic footprinting experiments in both E. coli (45) and H. influenzae (1).…”

“…Genetic footprinting is a three-step process: (i) random transposon mutagenesis of a large number of cells, (ii) competitive outgrowth of the mutagenized population under various selective conditions, and (iii) analysis of individual mutants surviving in the population using direct sequencing or various hybridization and PCR-based techniques. Various modifications of genetic footprinting have been recently applied to several microorganisms, including Mycoplasma genitalium and Mycoplasma pneumoniae (49), Pseudomonas aeruginosa (99), Helicobacter pylori (52), and Escherichia coli (7,45). Another version of this method, termed genomic analysis and mapping by in vitro transposition, has been developed for Haemophilus influenzae (1,75) and Streptococcus pneumoniae (1).…”

“…Genetic footprinting in E. coli by mini-Tn10 mutagenesis in vivo has been recently reported (7,45). In our systematic analysis of E. coli gene essentiality, we have chosen an approach based on the use of the "transposome," precut transposon DNA in a stable complex with modified Tn5 transposase (40,47).…”

Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

“…2). The two KpnI-BssSI fragments of pGT7 were ligated with an AatII-BamHI fragment of pAM34 (ATCC 77185, Hare et al 2001). Tn5Del8 is similar in structure to Tn5Del7, except that it contains a regulated origin of replication.…”

“…Transposition-based strategies have been developed recently for identifying essential genes (for review, see Judson and Mekalanos 2000a;Hamer et al 2001;Gerdes et al 2002). Conceptually, these methods are based on the fact that transposon insertion into a gene causes loss of gene function (gene knockout), and insertion into an essential gene is lethal to the organism and cannot be observed (Akerley et al 1998;Hare et al 2001). By generating large libraries with chromosomal transposon insertions, followed by sequencing or PCR analysis of inserts in surviving cells, it can be assumed that any gene that is not found to contain a transposon insertion is essential.…”