116
in antibiotic resistance (Gallagher et al. 2011), virulence (Fu et al. 2013) and persister cells formation (Molina-Quiroz et al. 2016; Shan et al. 2015).
The transposon mutant libraries of several bacterial species (Table 5.2) have been
constructed and screened for persister cells formation related genes in certain condition. A total of 5000 P. aeruginosa PA14 transposon insert mutants were screened
for persistence mutants under the pressure of ofloxacin treatment. This study has
identified 9 persister formation related genes (De Groote et al. 2009). Screening of
E. coli mutant library with ofloxacin found inactivation of phoU causes pansusceptibility to various antibiotics and decreased the persistence (Li and Zhang
2007). PhoU is a negative regulator of the Pho regulon and involved in phosphate
metabolism (Wanner 1990). Inactivation of homologue of PhoU in M. tuberculosis
also resulted in a defect in persistence (Shi and Zhang 2010). Screening the S. aureus
transposon insertion mutant library has identified 13 genes that contribute to persister cell formation, which are involved in oxidative phosphorylation, tricarboxylic
acid cycle (TCA) cycle, glycolysis, cell cycle, and ABC transporters (Wang et al.
2015). By screening of 576 insertion mutants of M. tuberculosis in isoniazid treated
mice persistence model, the disruption of cydC was found to decrease the persistence in isoniazid treated mice (Dhar and McKinney 2010). MT13, encoding a transcriptional regulatory factor in M. fortuitum (Parti et al. 2008), and pcaA encoding
a mycolic acid synthase in M. bovis BCG (Glickman et al. 2000) were identified as
persistence determining genes by insertion library screening.
Transposon mutant library based high throughput screening has contributed significantly to persistence genes discovery. However, it needs considerable amount of
labor and time to evaluate the phenotype of each mutant in the whole library. Instead,
transposon insertion site sequencing (Tn-seq), which combines the transposon
mutagenesis and next generation sequencing (NGS), provides a powerful and comprehensive approach for genome wide studies (Mazurkiewicz et al. 2006; van
Opijnen and Camilli 2013). By generating the transposon mutant library and culture
it under different stress conditions, the relative abundance of mutants can be tracked
via sequencing. The change in relative abundance of a certain mutation reflects that
the gene is critical (decrease in relative abundance) or deleterious (increase in relative abundance) under that specific condition (Goodman et al. 2011; van Opijnen
et al. 2009). Such studies led to the identification of thousands of persister genes in
different microorganisms, including E. coli (Molina-Quiroz et al. 2016; Moyed and
Bertrand 1983) and P. aeruginosa (Cameron et al. 2018).
5.4.2 Knockout and Overexpression Library Screening
The Keio deletion mutant library consist of 3985 defined single gene deletions of all
nonessential genes in E. coli K-12 (Baba et al. 2006; Yamamoto et al. 2009). Two
energy production genes, sucB (encoding the E2 subunit of the 2-oxoglutarate dehydrogenase complex) and ubiF (encoding 2-octaprenyl-3-methyl-6-methoxy1,4-benzoquinol oxygenase involved in ubiquinone biosynthesis) were identified as
X. Duan et al.
in antibiotic resistance (Gallagher et al. 2011), virulence (Fu et al. 2013) and persister cells formation (Molina-Quiroz et al. 2016; Shan et al. 2015).
The transposon mutant libraries of several bacterial species (Table 5.2) have been
constructed and screened for persister cells formation related genes in certain condition. A total of 5000 P. aeruginosa PA14 transposon insert mutants were screened
for persistence mutants under the pressure of ofloxacin treatment. This study has
identified 9 persister formation related genes (De Groote et al. 2009). Screening of
E. coli mutant library with ofloxacin found inactivation of phoU causes pansusceptibility to various antibiotics and decreased the persistence (Li and Zhang
2007). PhoU is a negative regulator of the Pho regulon and involved in phosphate
metabolism (Wanner 1990). Inactivation of homologue of PhoU in M. tuberculosis
also resulted in a defect in persistence (Shi and Zhang 2010). Screening the S. aureus
transposon insertion mutant library has identified 13 genes that contribute to persister cell formation, which are involved in oxidative phosphorylation, tricarboxylic
acid cycle (TCA) cycle, glycolysis, cell cycle, and ABC transporters (Wang et al.
2015). By screening of 576 insertion mutants of M. tuberculosis in isoniazid treated
mice persistence model, the disruption of cydC was found to decrease the persistence in isoniazid treated mice (Dhar and McKinney 2010). MT13, encoding a transcriptional regulatory factor in M. fortuitum (Parti et al. 2008), and pcaA encoding
a mycolic acid synthase in M. bovis BCG (Glickman et al. 2000) were identified as
persistence determining genes by insertion library screening.
Transposon mutant library based high throughput screening has contributed significantly to persistence genes discovery. However, it needs considerable amount of
labor and time to evaluate the phenotype of each mutant in the whole library. Instead,
transposon insertion site sequencing (Tn-seq), which combines the transposon
mutagenesis and next generation sequencing (NGS), provides a powerful and comprehensive approach for genome wide studies (Mazurkiewicz et al. 2006; van
Opijnen and Camilli 2013). By generating the transposon mutant library and culture
it under different stress conditions, the relative abundance of mutants can be tracked
via sequencing. The change in relative abundance of a certain mutation reflects that
the gene is critical (decrease in relative abundance) or deleterious (increase in relative abundance) under that specific condition (Goodman et al. 2011; van Opijnen
et al. 2009). Such studies led to the identification of thousands of persister genes in
different microorganisms, including E. coli (Molina-Quiroz et al. 2016; Moyed and
Bertrand 1983) and P. aeruginosa (Cameron et al. 2018).
5.4.2 Knockout and Overexpression Library Screening
The Keio deletion mutant library consist of 3985 defined single gene deletions of all
nonessential genes in E. coli K-12 (Baba et al. 2006; Yamamoto et al. 2009). Two
energy production genes, sucB (encoding the E2 subunit of the 2-oxoglutarate dehydrogenase complex) and ubiF (encoding 2-octaprenyl-3-methyl-6-methoxy1,4-benzoquinol oxygenase involved in ubiquinone biosynthesis) were identified as
X. Duan et al.