kanamycin, spectinomycin, or gentamicin [14–21]. However, sitedirected mutagenesis studies of Leptospira have remained very limited, such as to flaB, recA, and hemH genes in L. biflexa [22–24];
metY, metX, metW, and trpE in L. meyeri [15, 25]; and to a limited
number of genes in pathogenic L. interrogans species [26].
Alternatively, transposon-based mutagenesis is a powerful tool
for the study of bacterial physiology and pathogenesis through
random DNA insertions into a chromosome [27]. Himar1 transposon of the mariner family, which does not require host cofactors
to randomly insert into a TA dinucleotide within a chromosome,
has been used in a variety of prokaryotes, including the random Tn
library generation of Borrelia burgdorferi, the spirochete that is the
etiologic agent of Lyme disease [28–34]. Recently, Himar1 has
been adapted for use with Leptospira to create a large number of Tn
mutants within saprophytic as well as pathogenic species, in which
the latter has proven more challenging to generate [16–18]. Previous research has developed Himar1 insertion into Leptospira by
conjugation [17]. As an example, conjugative plasmid pCjTKS1
can deliver Himar1 more efficiently than electroporation, with a
DNA transfer frequency ranging from 1 Â 10
À6 to 8.5 Â 10
À8
transconjugants/recipient cell in L. biflexa and L. interrogans,
respectively [17].
Here, we describe the methodology to generate random transposon mutants in the saprophyte L. biflexa via conjugation through
E. coli β2163 as a donor cell harboring pCjTKS1-derived conjugative plasmid pSW29T-TKS2. Extensive libraries of saprophytic and
pathogenic Leptospira Tn mutants created by this method could be
used for studying the phenotypes of diverse Tn gene mutations
including but not limited to amino acid biosynthesis, motility, iron
acquisition [16, 35], virulence [36, 37], and sensitivity to ethidium
bromide [38], among others. Additionally, the generation of Tn
libraries could foster and facilitate collaborations between laboratories investigating different aspects of Leptospira physiology
through sharing the Tn mutant libraries. For example, collaborations can valuably reduce the substantial resource and labor expenditure associated with the generation, sequencing, and upkeep of
the library. Further, such collaborations foster a positive research
and learning environment between laboratories, ultimately supporting research to transcend institutional borders.
2 Materials
Use only sterilized/autoclaved glassware for all procedures. Set
aside specific glassware for EMJH preparation and Leptospira
work. Do not use general glassware supplies as these may be contaminated with other chemicals to which Leptospira are sensitive.
78
Christopher J. Pappas et al.
metY, metX, metW, and trpE in L. meyeri [15, 25]; and to a limited
number of genes in pathogenic L. interrogans species [26].
Alternatively, transposon-based mutagenesis is a powerful tool
for the study of bacterial physiology and pathogenesis through
random DNA insertions into a chromosome [27]. Himar1 transposon of the mariner family, which does not require host cofactors
to randomly insert into a TA dinucleotide within a chromosome,
has been used in a variety of prokaryotes, including the random Tn
library generation of Borrelia burgdorferi, the spirochete that is the
etiologic agent of Lyme disease [28–34]. Recently, Himar1 has
been adapted for use with Leptospira to create a large number of Tn
mutants within saprophytic as well as pathogenic species, in which
the latter has proven more challenging to generate [16–18]. Previous research has developed Himar1 insertion into Leptospira by
conjugation [17]. As an example, conjugative plasmid pCjTKS1
can deliver Himar1 more efficiently than electroporation, with a
DNA transfer frequency ranging from 1 Â 10
À6 to 8.5 Â 10
À8
transconjugants/recipient cell in L. biflexa and L. interrogans,
respectively [17].
Here, we describe the methodology to generate random transposon mutants in the saprophyte L. biflexa via conjugation through
E. coli β2163 as a donor cell harboring pCjTKS1-derived conjugative plasmid pSW29T-TKS2. Extensive libraries of saprophytic and
pathogenic Leptospira Tn mutants created by this method could be
used for studying the phenotypes of diverse Tn gene mutations
including but not limited to amino acid biosynthesis, motility, iron
acquisition [16, 35], virulence [36, 37], and sensitivity to ethidium
bromide [38], among others. Additionally, the generation of Tn
libraries could foster and facilitate collaborations between laboratories investigating different aspects of Leptospira physiology
through sharing the Tn mutant libraries. For example, collaborations can valuably reduce the substantial resource and labor expenditure associated with the generation, sequencing, and upkeep of
the library. Further, such collaborations foster a positive research
and learning environment between laboratories, ultimately supporting research to transcend institutional borders.
2 Materials
Use only sterilized/autoclaved glassware for all procedures. Set
aside specific glassware for EMJH preparation and Leptospira
work. Do not use general glassware supplies as these may be contaminated with other chemicals to which Leptospira are sensitive.
78
Christopher J. Pappas et al.