This work was supported by a Veterans Affairs Merit Award (to D.A.H.) and a National Institute of Health grant R01 AI 034431 (to D.A.H.).

<ol>
	<li>Murray, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+transposon+mutagenesis+in+pathogenic+Leptospira+species.">Genome-wide transposon mutagenesis in pathogenic Leptospira species.</a> Infect Immun. 77 (2), 810-816 (2009).</li><li>Adler, B., Lo, M., Seemann, T., Murray, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+leptospirosis:+the+influence+of+genomics.">Pathogenesis of leptospirosis: the influence of genomics.</a> Vet Microbiol. 153 (1-2), 73-81 (2011).</li><li>Marcsisin, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+high-throughput+screen+to+identify+Leptospira+mutants+unable+to+colonize+the+carrier+host+or+cause+disease+in+the+acute+model+of+infection.">Use of a high-throughput screen to identify Leptospira mutants unable to colonize the carrier host or cause disease in the acute model of infection.</a> J Med Microbiol. 62, 1601-1608 (2013).</li><li>van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn-seq:+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms.">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nat Methods. 6 (10), 767-772 (2009).</li><li>van Opijnen, T., Lazinski, D. W., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+fitness+and+genetic+interactions+determined+by+Tn-seq,+a+high-throughput+massively+parallel+sequencing+method+for+microorganisms.">Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms.</a> Curr Protoc Microbiol. 36, 1E.3.1-1E.3.24 (2015).</li><li>Troy, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+barriers+to+Borrelia+burgdorferi+dissemination+during+infection+using+massively+parallel+sequencing.">Understanding barriers to Borrelia burgdorferi dissemination during infection using massively parallel sequencing.</a> Infect Immun. 81 (7), 2347-2357 (2013).</li><li>Wunder, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+reveals+rapid+dissemination+of+Leptospira+interrogans+after+intraperitoneal+and+conjunctival+inoculation+of+hamsters.">Real-time PCR reveals rapid dissemination of Leptospira interrogans after intraperitoneal and conjunctival inoculation of hamsters.</a> Infect Immun. 84 (7), 2105-2115 (2016).</li><li>Lourdault, K., Aviat, F., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+quantitative+real-time+PCR+for+studying+the+dissemination+of+Leptospira+interrogans+in+the+guinea+pig+infection+model+of+leptospirosis.">Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.</a> J Med Microbiol. 58, 648-655 (2009).</li><li>Coutinho, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+Leptospira+interrogans+infection+in+hamsters+after+intradermal+and+subcutaneous+challenge.">Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge.</a> PLoS Negl Trop Dis. 8 (11), e3307 (2014).</li><li>Athanazio, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rattus+norvegicus+as+a+model+for+persistent+renal+colonization+by+pathogenic+Leptospira+interrogans.">Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans.</a> Acta Trop. 105 (2), 176-180 (2008).</li><li>Ratet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+bioluminescent+Leptospira+interrogans+in+mice+reveals+renal+colonization+as+a+stealth+escape+from+the+blood+defenses+and+antibiotics.">Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics.</a> PLoS Negl Trop Dis. 8 (12), e3359 (2014).</li><li>Gutierrez, M. G., Yoder-Himes, D. R., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+virulence+factors+required+for+respiratory+melioidosis+using+Tn-seq+mutagenesis.">Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis.</a> Front Cell Infect Microbiol. 5 (78), (2015).</li><li>Gawronski, J. D., Wong, S. M., Giannoukos, G., Ward, D. V., Akerley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+insertion+mutants+within+libraries+by+deep+sequencing+and+a+genome-wide+screen+for+Haemophilus+genes+required+in+the+lung.">Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.</a> Proc Natl Acad Sci U S A. 106 (38), 16422-16427 (2009).</li><li>Gallagher, L. A., Shendure, J., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+identification+of+resistance+functions+in+Pseudomonas+aeruginosa+using+Tn-seq.">Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq.</a> MBio. 2 (1), (2011).</li><li>Slamti, L., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+library+of+random+mutants+in+the+spirochete+Leptospira+biflexa+using+a+mariner+transposon.">Construction of a library of random mutants in the spirochete Leptospira biflexa using a mariner transposon.</a> Methods Mol Biol. 859, (2012).</li><li>Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugative+transfer+between+Escherichia+coli+and+Leptospira+spp.+as+a+new+genetic+tool.">Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool.</a> Appl Environ Microbiol. 74 (1), 319-322 (2008).</li><li>Lourdault, K., Matsunaga, J., Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+parallel+sequencing+to+measure+fitness+of+Leptospira+interrogans+transposon+insertion+mutants+during+acute+infection.">High-throughput parallel sequencing to measure fitness of Leptospira interrogans transposon insertion mutants during acute infection.</a> PLoS Negl Trop Dis. 10 (11), e0005117 (2016).</li><li>Ellinghausen, H. C., McCullough, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrition+of+Leptospirapomona+and+growth+of+13+other+serotypes:+Fractionation+of+oleic+albumin+complex+and+a+medium+of+bovine+albumin+and+polysorbate+80.">Nutrition of Leptospirapomona and growth of 13 other serotypes: Fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80.</a> Am J Vet Res. 26, 45-51 (1965).</li><li>Johnson, R. C., Harris, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+pathogenic+and+saprophytic+letospires.+I.+Growth+at+low+temperatures.">Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures.</a> J Bacteriol. 94 (1), 27-31 (1967).</li><li>Demarre, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+mobilizable+suicide+plasmids+based+on+broad+host+range+R388+plasmid+(IncW)+and+RP4+plasmid+(IncPa)+conjugative+machineries+and+their+cognate+Escherichia+coli+host+strains.">A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPa) conjugative machineries and their cognate Escherichia coli host strains.</a> Res Microbiol. 156 (2), 245-255 (2005).</li><li>Vallenet, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroScope:+a+platform+for+microbial+genome+annotation+and+comparative+genomics.">MicroScope: a platform for microbial genome annotation and comparative genomics.</a> Database (Oxford). , bap021 (2009).</li><li>Goodman, A. L., Wu, M., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+microbial+fitness+determinants+by+insertion+sequencing+using+genome-wide+transposon+mutant+libraries.">Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries.</a> Nat. Protocols. 6 (12), 1969-1980 (2011).</li><li>Miller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spirochetes in body fluids and tissues manual of investigative methods. , 22-23 (1971).</li><li>Ristow, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OmpA-like+protein+Loa22+is+essential+for+leptospiral+virulence.">The OmpA-like protein Loa22 is essential for leptospiral virulence.</a> PLoS Pathog. 3 (7), (2007).</li><li>Croda, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+in+pathogenic+Leptospira+species:+disruption+of+the+LigB+gene+does+not+affect+virulence+in+animal+models+of+leptospirosis.">Targeted mutagenesis in pathogenic Leptospira species: disruption of the LigB gene does not affect virulence in animal models of leptospirosis.</a> Infect Immun. 76 (12), 5826-5833 (2008).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hamster+model+of+leptospirosis.">Hamster model of leptospirosis.</a> Curr Protoc Microbiol. 12, 2 (2006).</li><li>Giardine, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy:+a+platform+for+interactive+large-scale+genome+analysis.">Galaxy: a platform for interactive large-scale genome analysis.</a> Genome Res. 15 (10), 1451-1455 (2005).</li><li>Goecks, J., Nekrutenko, A., Taylor, J., Galaxy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy:+a+comprehensive+approach+for+supporting+accessible,+reproducible,+and+transparent+computational+research+in+the+life+sciences.">Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.</a> Genome Biol. 11 (8), R86 (2010).</li><li>Blankenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy:+a+web-based+genome+analysis+tool+for+experimentalists.">Galaxy: a web-based genome analysis tool for experimentalists.</a> Curr Protoc Mol Biol. (Chapter 19), 11-21 (2010).</li><li>Afgan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Galaxy+platform+for+accessible,+reproducible+and+collaborative+biomedical+analyses:+2016+update.">The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update.</a> Nucleic Acids Res. 8 (44), (2016).</li><li>McCoy, K. M., Antonio, M. L., van Opijnen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAGenTA;+a+Galaxy+implemented+tool+for+complete+Tn-Seq+analysis+and+data+visualization.">MAGenTA; a Galaxy implemented tool for complete Tn-Seq analysis and data visualization.</a> Bioinformatics. , 1367 (2017).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10 (3), R25 (2009).</li><li>Kamp, H. D., Patimalla-Dipali, B., Lazinski, D. W., Wallace-Gadsden, F., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+fitness+landscapes+of+Vibrio+cholerae+at+important+stages+of+its+life+cycle.">Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle.</a> PLoS Pathog. 9 (12), e1003800 (2013).</li><li>Poggi, D., Oliveira de Giuseppe, P., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+markers+for+genetic+manipulations+of+Leptospira+spp.">Antibiotic resistance markers for genetic manipulations of Leptospira spp.</a> Appl Environ Microbiol. 76 (14), 4882-4885 (2010).</li><li>van Opijnen, T., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+insertion+sequencing:+a+new+tool+for+systems-level+analysis+of+microorganisms.">Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.</a> Nat Rev Microbiol. 11 (7), 435-442 (2013).</li><li>Pappas, C. J., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+gene+expression+in+Leptospira+spp.+by+Transcription+activator-like+effectors+demonstrates+a+potential+role+for+LigA+and+LigB+in+Leptospira+interrogans+virulence.">Control of gene expression in Leptospira spp. by Transcription activator-like effectors demonstrates a potential role for LigA and LigB in Leptospira interrogans virulence.</a> Appl Environ Microbiol. 81 (22), 7888-7892 (2015).</li><li>Koskiniemi, S., Sun, S., Berg, O. G., Andersson, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection-driven+gene+loss+in+bacteria.">Selection-driven gene loss in bacteria.</a> PLoS Genet. 8 (6), e1002787 (2012).</li><li>Troy, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Tn-seq+analysis+of+carbohydrate+utilization+and+vertebrate+infectivity+of+Borrelia+burgdorferi.">Global Tn-seq analysis of carbohydrate utilization and vertebrate infectivity of Borrelia burgdorferi.</a> Mol Microbiol. 101 (6), 1003-1023 (2016).</li><li>Ramsey, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+genetic+screen+identifies+previously+uncharacterized+Borrelia+burgdorferi+genes+important+for+resistance+against+reactive+oxygen+and+nitrogen+species.">A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.</a> PLoS Negl Trop Dis. 13 (2), e1006225 (2017).</li></ol>

The authors have nothing to disclose.

Although results from our pilot experiment for hamster challenged intraperitoneally with 42 L. interrogans mutants are presented17, we expect that larger pools of mutants can be screened by Tn-Seq. Because the frequency of transconjugants is low (100-200 transconjugants/mating), several matings are necessary to generate a sufficient number of mutants for large Tn-Seq experiments. Maintaining a large number of mutants in liquid cultures presents logistical challenges that must be addressed. Cultures can be incubated in deep 96-well plates. Culture densities can be monitored by optical density readings in a spectrophotometer set to 420 nm. The determination of the number of animals to use depends in part on the size of the input pool and must be determined by power analysis. For large-scale experiments, we recommend that the power analysis be performed in consultation with a biostatistician.
Bottlenecks can impact the recovery of random mutants from the output pool. Although severe bottlenecks were not observed in our pilot study17 (Figure 7), large-scale experiments may be affected by random loss of mutants. Increasing the challenge dose by increasing the number of cells per mutant in the input pool will minimize the likelihood of bottlenecks35. The newly published Galaxy tool MAGenTA provides the necessary tool to assess bottlenecks and fitness31.
Tn-Seq experiments planned for an alternative route of infection, other Leptospira strains, or other rodent models require additional considerations. If the kinetics of infection in the host species is not known or is not continuously exponential during the course of infection within each tissue to be examined, DNA should be obtained from culturing of the tissues rather than directly from the tissues. This additional step will minimize overestimates of mutant fitness due to detection of DNA from dead spirochetes. If the output pool is obtained from culturing the tissues, the input pool should be cultured to address effects of in vitro growth. We also recommend a pilot experiment with a small number of mutants to determine whether bottlenecks will be a concern and to aid the power analysis for large-scale experiments.
Confirmation of altered fitness as determined by Tn-Seq will require testing of individual mutants in the animal model. Currently, each mutant must be sequenced individually prior to Tn-Seq to identify the mutant carrying the insertion in the gene of interest. However, if allele replacement in pathogenic Leptospira becomes easy, sequencing of individual mutants will no longer be necessary. Alternatively, transcription activator-like effectors have been successfully used to diminish lig genes expression in L. interrogans36. This technique could be used to down-regulate genes disrupted in mutants with altered fitness identified by Tn-Seq.
When interpreting the Tn-Seq data, interactions between bacteria should to be taken into account. It is theoretically possible that a decrease in fitness could be due to competition between mutants rather than a direct effect of the transposon insertion. Additionally, an absence of change in in vivo fitness of a mutant in a pool could be due to cooperation between mutants. For example, a mutant that fails to produce an essential factor could be complemented intercellularly by production of the factor by other mutants in the pool. We observed an increase in fitness for several mutants, which may be a consequence of the reduced metabolic burden of no longer synthesizing proteins that are not essential for growth, as shown for Salmonella enterica37.
This protocol can be used to identify genes involved in metabolism or survival under stressful conditions in vitro 38,39. For example, growing Leptospira in different conditions like high sodium chloride concentration, limited iron, and acidic pH could identify genes responsible for acidic survival or stress resistance. These in vitro experiments can also be performed with saprophytic strains such as L. biflexa strain Patoc I because this Tn-Seq method can be applied to all sequenced Leptospira strains.

Identification of virulence genes for some bacteria, such as Leptospira spp., is difficult because of the limited number of genetic tools available. One commonly used approach is the creation of a collection of mutants by random transposon mutagenesis followed by the identification of the insertion site in each mutant and virulence testing of individual transposon mutants in an animal model. This approach is time-consuming, expensive, and requires a large number of animals.
When random mutagenesis was first developed for the pathogen Leptospira interrogans, genes involved in virulence were identified by testing individual mutants in an animal model1. Mutants were selected based on criteria such as their potential roles in signaling or motility or their predicted outer membrane or surface location. As the majority of leptospiral genes encode hypothetical proteins of unknown function2, selecting mutants based on these criteria limits the ability to discover novel leptospiral virulence genes.
More recently, pools of L. interrogans transposon mutants were screened for infectivity in the hamster and mouse models3. Each animal was challenged with a pool of up to 10 mutants. Infectivity of a mutant was scored as positive if it was detected by PCR of cultures obtained from blood and kidney. PCR testing was laborious because it required an individual PCR reaction for each mutant in the pool. Because the frequency of each mutant in the cultures was not quantified, the approach was biased towards identification of highly attenuated mutants.
We describe a transposon sequencing (Tn-Seq) technique, as a strategy to more efficiently screen for virulence genes. Tn-seq consists of the creation of a library of mutants by transposon mutagenesis followed by massive parallel sequencing4,5,6. Briefly, transposon mutants are pooled, inoculated into animals, and later recovered from different organs (output pools). The DNA from the output pools is extracted and digested with restriction enzymes or sheared by sonication. Two rounds of PCR targeting the junctions of the transposon insertion sites are performed. This step enables the addition of the adaptors necessary for the sequencing. The resulting PCR products are analyzed by high-throughput sequencing to identify the transposon insertion site of each mutant of the pool along with their relative abundance, which is compared to the initial composition of the pool of mutant.
The primary advantage of this approach is the ability to simultaneously screen a large number of mutants with a small number of animals. Tn-Seq does not require the prior knowledge of the transposon insertion sites which increases the chances of discovering new Leptospira-specific genes involved in virulence with less time and greater efficiency. Because leptospiral burden in tissues is relatively high in rodent models susceptible to lethal infection (typically 104 to 108 bacteria/g of tissue)7,8,9 as well as in reservoir hosts10,11, tissues can be directly analyzed without the need to culture, reducing biases due to in vitro growth.
In Tn-Seq studies with most bacterial pathogens described to date, the high frequency of insertional mutagenesis allowed infection with large pools containing mutants collectively having multiple closely-spaced transposon insertions within every gene4,12,13,14. Tn-Seq has also been developed for a bacterium for which the mutagenesis frequency is much lower6. With Leptospira, a library of transposon mutants can be generated by introducing the transposon on a mobilizable plasmid by conjugation as described by Slamti et al15. However, the frequency of transposon mutagenesis of L. interrogans is low. When the Himar1 transposon was introduced on a conjugative plasmid, the transconjugant frequency was reported to be only 8.5 x 10-8 per recipient cell with the Lai strain of L. interrogans16 and is likely to be similarly poor with most other strains of L. interrogans. The protocol described here is in part based on that developed for Borrelia burgdorferi, in which the frequency of transposon insertional mutagenesis is also low6.
For our pilot experiment with the protocol17, we conducted transposon mutagenesis with L. interrogans serovar Manilae strain L495 because of the success of other groups in isolating transposon insertion mutants in the strain along with its low LD50 (lethal dose) for virulence1. We screened 42 mutants by Tn-Seq and identified several mutant candidates defective in virulence, including two with insertions in a candidate adenylate cyclase gene. Individual testing of the two mutants in hamsters confirmed that they were deficient in virulence17.

<table><tbody><tr><td>Kanamycin sulfate from &lt;em&gt;Streptomyces kanamyceticus&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>K4000</td><td></td></tr><tr><td>2,6-diaminopimelic acid</td><td>Sigma-Aldrich</td><td>D1377</td><td></td></tr><tr><td>Spectinomycin dihydrochloride pentahydrate</td><td>Sigma-Aldrich</td><td>S4014</td><td></td></tr><tr><td>Axio Lab A1 microscope with a darkfieldcondenser</td><td>Zeiss</td><td>490950-001-000</td><td></td></tr><tr><td>DNeasy blood and tissue kit</td><td>Qiagen</td><td>69504/69506</td><td></td></tr><tr><td>MinElute PCR Purification</td><td>Qiagen</td><td>28004/28006</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104/28106</td><td></td></tr><tr><td>Model 505 Sonic Dismembrator</td><td>Fisher Scientific</td><td>FB-505</td><td></td></tr><tr><td>2.5&quot; Cup horn</td><td>Fisher Scientific</td><td>FB-4625</td><td></td></tr><tr><td>Bead Ruptor 24</td><td>Omni International</td><td>19-010</td><td>Step 3.1.2.4</td></tr><tr><td>Terminal deoxynucleotidyl transferase</td><td>Promega</td><td>M828C</td><td></td></tr><tr><td>Master mix Phusion</td><td>Thermo Scientific</td><td>F531</td><td>Preparation of genomic libraries, step 3.4.</td></tr><tr><td>DreamTaq Master Mix</td><td>Thermo Scientific</td><td>K9011/K9012</td><td>Identification of the transposon insertion site, step 1.2.</td></tr><tr><td>dCTP</td><td>Thermo Scientific</td><td>R0151</td><td></td></tr><tr><td>ddCTP</td><td>Affymetrix/ USBProducts</td><td>77112</td><td></td></tr><tr><td>T100 Thermal cycler</td><td>BioRad</td><td>1861096</td><td></td></tr><tr><td>Qubit 2.0 fluorometer</td><td>Invitrogen</td><td>Q32866</td><td>step 3.6.</td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Invitrogen</td><td>Q32851/Q32854</td><td>step 3.6.</td></tr><tr><td>Qubit assay tubes</td><td>life technologies</td><td>Q32856</td><td>step 3.6.</td></tr><tr><td>PBS pH 7.2 (1X)</td><td>Gibco</td><td>20012-027 20012-050</td><td></td></tr><tr><td>Disposable scalpel No10</td><td>Feather</td><td>2975#10</td><td></td></tr><tr><td>Plastic K&lt;sub&gt;2&lt;/sub&gt; EDTA 2 ml tubes</td><td>BD vacutainer</td><td>367841</td><td></td></tr><tr><td>syringe U-100 with 26G x &amp;frac12;&amp;rdquo; needle</td><td>BD vacutainer</td><td>329652</td><td>IP challenge, step 2.2.1.</td></tr><tr><td>3 mL Luer-Lok tip syringe</td><td>BD vacutainer</td><td>309657</td><td>Cardiac puncture, step 2.4.2.</td></tr><tr><td>25G X 5/8&amp;rdquo; needle</td><td>BD vacutainer</td><td>305901</td><td>Cardiac puncture, step 2.4.2.</td></tr><tr><td>25 mm fritted glass base with stopper</td><td>EMD Millipore</td><td>XX1002502</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>25 mm aluminum spring clamp</td><td>EMD Millipore</td><td>XX1002503</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>15 ml borosilcate glass funnel</td><td>EMD Millipore</td><td>XX1002514</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>125 ml side-arm Erlenmeyer flask</td><td>EMD Millipore</td><td>XX1002505</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm)</td><td>EMD Millipore</td><td>VVLP02500</td><td></td></tr></tbody></table>

high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

high-throughput-parallel-sequencing-to-measure-fitness-leptospira

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.9K Views.  Veterans Affairs Greater Los Angeles Healthcare System. Hier beschrijven we een techniek die transposon mutagenese met hoge gegevensdoorvoer sequencing combineert te identificeren en kwantificeren van transposon leptospiral mutanten in weefsels na een uitdaging van hamsters. Dit protocol kan worden gebruikt voor scherm mutanten voor overleving en verspreiding in dieren en kan ook worden toegepast op in vitro studies.

Video: High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Genetics

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Medicine

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain &#946;2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Bioengineering

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Biology

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Samenvatting

Meer video's verkennen