Insertions of Mini-Tn10 Transposon T-POP in Salmonella enterica sv. typhi

Abstract

We have mutagenized a clinical strain of Salmonella enterica sv. typhi with mini-transposon Tn10dTet (T-POP) to obtain conditional lethal (tetracycline-dependent) mutants with T-POP insertions upstream of essential genes. Generalized transducing phage P22 was used to introduce T-POP from a S. typhimurium donor into a S. typhi recipient. Chromosomal DNA was purified from the mutagenized donor strains, fragmented, and then electroporated into S. typhi to backcross the original T-POP insertions. Four tetracycline-dependent mutants with two distinct terminal phenotypes were found among 1700 mutants with T-POP insertions. When grown in the absence of tetracycline, two of the four tetracycline-dependent mutants arrest at a late stage in the cell cycle, can be rescued by outgrowth in media with tetracycline, and define a reversible checkpoint late in the cell cycle. One of these insertions creates an operon fusion with a gene, yqgF, that is conserved among gram-negative bacteria and likely encodes an essential Holliday junction resolvase. T-POP insertions can be used not only to identify essential S. typhi genes but also to reveal novel phenotypes resulting from the depletion of their products. Thus, the construction of S. typhi mutants with deletions generalized transducing phage for S. typhi has yet to be of nonessential genes and assays of their phenotypes in found. Although P22 can adsorb to S. typhi and inject human cells will identify many new virulence determiits DNA into a S. typhi recipient, P22 cannot develop nants. However, because one can make deletions only lytically in S. typhi. Therefore, P22 cannot be used to of nonessential genes, this PCR-based method for gene disruption does not yield information about the roles that essential genes play in virulence.

Mycobacterium tuberculosis RuvX is a Holliday junction resolvase formed by dimerisation of the monomeric YqgF nuclease domain

“…This new family of putative HJRs is typified by E. coli YqgF protein. The yqgF gene is highly conserved among bacterial genomes (Aravind et al ., ; Ponting, ) and, unlike RuvC, is essential for growth of many bacteria, including E. coli (Freiberg et al ., ), Mycobacterium tuberculosis (Sassetti et al ., ), and Salmonella enterica serovar typhimurium (Hidalgo et al ., ). Various studies have found notable structural similarities in the folds of E. coli RuvC and YqgF proteins (Supporting Information Fig.…”

Insights from Comparative Genomics of the Genus Salmonella

“…An alternative approach was published in 2004 (based on previously developed techniques) to identify larger numbers of essential genes, by insertion of conditional lethal mutations into random gene fragments in a S. typhimurium strain [7]. The conditional switch used here was growth temperature, while tetracycline-dependent expression was used by others [8], although they only reported indings for four essential genes. A few years later, transposon (Tn) mutagenesis combined with high-throughput sequencing became available and this was applied to S. enterica strains [9][10][11][12].…”

“…Exposure to low pH has also been tested [8]. Moreover, even 'essential' genes can often endure a transposon insertion without complete loss of function.…”

Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase

“…In an attempt to make use of these properties, a Tn10-based system was developed previously (28); however, this transposon has the disadvantage of nonrandom insertion, leading to mutational hotspots (1). Furthermore, a Tn10 variant in which the tetracycline resistance gene tetA was present has been employed (20)(21)(22). As a result, tetracycline-induced transcription must proceed from P A through the resistance gene before reaching the target gene downstream of the insertion site.…”