found this is unnecessary as we still get good sequencing data,
even if quality becomes low from enzymatic purification.
47. We typically use either NCBI BLAST (https://blast.ncbi.nlm.
nih.gov/Blast.cgi) or Genoscope (http://www.genoscope.cns.
fr/agc/microscope/home/index.php). Either website yields
similar results. One advantage of using Genoscope is that
many bioinformatic programs are also available within the
program for initial characterization of the disrupted gene that
is identified by BLAST analysis.
48. Transposon may have inserted in same or reverse orientation as
disrupted gene since the FRT sites in pSW29T-TKS2 are not in
tandem. This allows the transposon to become inverted.
Acknowledgments
This work was supported by NIH grant 1R01AI132818 (M.A.M.)
and the Manhattanville College Faculty Research Fund (C.J.P.).
References
1. Haake DA, Levett PN (2015) Leptospirosis in
humans. Curr Top Microbiol Immunol
387:65–97. https://doi.org/10.1007/978-3662-45059-8_5
2. Adler B, de la Pena Moctezuma A (2010) Leptospira and leptospirosis. Vet Microbiol
140:287–296.
https://doi.org/10.1016/j.
vetmic.2009.03.012
3. Chiriboga J, Barragan V, Arroyo G, Sosa A,
Birdsell DN, Espan ˜a K, Mora A, Espı ´n E,
Mejı ´a ME, Morales M, Pinargote C,
Gonzalez M, Hartskeerl R, Keim P, Bretas G,
Eisenberg JNS, Trueba G (2015) High prevalence of intermediate Leptospira spp. DNA in
febrile humans from urban and rural ecuador.
Emerg Infect Dis 21:2141–2147. https://doi.
org/10.3201/eid2112.140659
4. Costa F, Hagan JE, Calcagno J, Kane M,
Torgerson P, Martinez-Silveira MS, Stein C,
Abela-Ridder B, Ko AI (2015) Global morbidity and mortality of leptospirosis: a systematic
review. PLoS Negl Trop Dis 9:e0003898.
https://doi.org/10.1371/journal.pntd.
0003898
5. Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14:296–326. https://doi.org/10.
1128/CMR.14.2.296-326.2001
6. Evangelista KV, Coburn J (2010) Leptospira as
an emerging pathogen: a review of its biology,
pathogenesis and host immune responses.
Future Microbiol 5(9):1413–1425
7. De Brito T, da Silva AMG, Abreu PAE (2018)
Pathology and pathogenesis of human leptospirosis: a commented review. Rev Inst Med
Trop Sao Paulo 60:e23
8. Guernier V, Goarant C, Benschop J, Lau CL
(2018) A systematic review of human and animal leptospirosis in the Pacific Islands reveals
pathogen and reservoir diversity. PLoS Negl
Trop Dis 12(5):e0006503. https://doi.org/
10.1371/journal.pntd.0006503
9. Wang S, Stobart Gallagher MA, Dunn N
(2020) Leptospirosis (Weil Disease). In: StatPearls. Treasure Island (FL): StatPearls
Publishing
10. Yelton DB, Cohen RA (1986) Analysis of
cloned DNA from Leptospira biflexa serovar
patoc which complements a deletion of the
Escherichia coli trpE gene. J Bacteriol 165
(1):41–46. https://doi.org/10.1128/jb.165.
1.41-46.1986
11. Stamm LV, Parrish EA, Gherardini FC (1991)
Cloning of the recA gene from a free-living
leptospire and distribution of RecA-like protein
among spirochetes. Appl Environ Microbiol 57
(1):183–189
12. Baril C, Richaud C, Fournie E, Baranton G,
Saint GI (1992) Cloning of dapD, aroD and
asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd
gene. J Gen Microbiol 138(1):47–53
94
Christopher J. Pappas et al.
even if quality becomes low from enzymatic purification.
47. We typically use either NCBI BLAST (https://blast.ncbi.nlm.
nih.gov/Blast.cgi) or Genoscope (http://www.genoscope.cns.
fr/agc/microscope/home/index.php). Either website yields
similar results. One advantage of using Genoscope is that
many bioinformatic programs are also available within the
program for initial characterization of the disrupted gene that
is identified by BLAST analysis.
48. Transposon may have inserted in same or reverse orientation as
disrupted gene since the FRT sites in pSW29T-TKS2 are not in
tandem. This allows the transposon to become inverted.
Acknowledgments
This work was supported by NIH grant 1R01AI132818 (M.A.M.)
and the Manhattanville College Faculty Research Fund (C.J.P.).
References
1. Haake DA, Levett PN (2015) Leptospirosis in
humans. Curr Top Microbiol Immunol
387:65–97. https://doi.org/10.1007/978-3662-45059-8_5
2. Adler B, de la Pena Moctezuma A (2010) Leptospira and leptospirosis. Vet Microbiol
140:287–296.
https://doi.org/10.1016/j.
vetmic.2009.03.012
3. Chiriboga J, Barragan V, Arroyo G, Sosa A,
Birdsell DN, Espan ˜a K, Mora A, Espı ´n E,
Mejı ´a ME, Morales M, Pinargote C,
Gonzalez M, Hartskeerl R, Keim P, Bretas G,
Eisenberg JNS, Trueba G (2015) High prevalence of intermediate Leptospira spp. DNA in
febrile humans from urban and rural ecuador.
Emerg Infect Dis 21:2141–2147. https://doi.
org/10.3201/eid2112.140659
4. Costa F, Hagan JE, Calcagno J, Kane M,
Torgerson P, Martinez-Silveira MS, Stein C,
Abela-Ridder B, Ko AI (2015) Global morbidity and mortality of leptospirosis: a systematic
review. PLoS Negl Trop Dis 9:e0003898.
https://doi.org/10.1371/journal.pntd.
0003898
5. Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14:296–326. https://doi.org/10.
1128/CMR.14.2.296-326.2001
6. Evangelista KV, Coburn J (2010) Leptospira as
an emerging pathogen: a review of its biology,
pathogenesis and host immune responses.
Future Microbiol 5(9):1413–1425
7. De Brito T, da Silva AMG, Abreu PAE (2018)
Pathology and pathogenesis of human leptospirosis: a commented review. Rev Inst Med
Trop Sao Paulo 60:e23
8. Guernier V, Goarant C, Benschop J, Lau CL
(2018) A systematic review of human and animal leptospirosis in the Pacific Islands reveals
pathogen and reservoir diversity. PLoS Negl
Trop Dis 12(5):e0006503. https://doi.org/
10.1371/journal.pntd.0006503
9. Wang S, Stobart Gallagher MA, Dunn N
(2020) Leptospirosis (Weil Disease). In: StatPearls. Treasure Island (FL): StatPearls
Publishing
10. Yelton DB, Cohen RA (1986) Analysis of
cloned DNA from Leptospira biflexa serovar
patoc which complements a deletion of the
Escherichia coli trpE gene. J Bacteriol 165
(1):41–46. https://doi.org/10.1128/jb.165.
1.41-46.1986
11. Stamm LV, Parrish EA, Gherardini FC (1991)
Cloning of the recA gene from a free-living
leptospire and distribution of RecA-like protein
among spirochetes. Appl Environ Microbiol 57
(1):183–189
12. Baril C, Richaud C, Fournie E, Baranton G,
Saint GI (1992) Cloning of dapD, aroD and
asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd
gene. J Gen Microbiol 138(1):47–53
94
Christopher J. Pappas et al.