Transposon-insertion sequencing as a tool to elucidate bacterial colonization factors in a Burkholderia gladioli symbiont of Lagria villosa beetles

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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 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 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.

OriginalsprogEngelsk
Artikelnummere62843
TidsskriftJournal of Visualized Experiments
Vol/bind174
Antal sider21
ISSN1940-087X
DOI
StatusUdgivet - 2021

Bibliografisk note

Funding Information:
We are thankful to Junbeom Lee for providing the E. coli WM3064+pRL27 strain for conjugation and guidance in the procedure, Kathrin H?ffmeier for helping with troubleshooting during mutant library generation, and Prof. Andr? Rodrigues for supporting insect collection and permit acquisition. We also thank Rebekka Janke and Dagmar Klebsch for support in the collection and rearing of the insects. We acknowledge the Brazilian authorities for granting the following permits for access, collection, and export of insect specimens: SISBIO authorization Nr. 45742-1, 45742-7 and 45742-10, CNPq process n? 01300.004320/2014-21 and 01300.0013848/2017-33, IBAMA Nr. 14BR016151DF and 20BR035212/DF). This research was supported by funding from the German Science Foundation (DFG) Research Grants FL1051/1-1 and KA2846/6-1.

Funding Information:
We are thankful to Junbeom Lee for providing the E. coli WM3064+pRL27 strain for conjugation and guidance in the procedure, Kathrin Hüffmeier for helping with troubleshooting during mutant library generation, and Prof. André Rodrigues for supporting insect collection and permit acquisition. We also thank Rebekka Janke and Dagmar Klebsch for support in the collection and rearing of the insects. We acknowledge the Brazilian authorities for granting the following permits for access, collection, and export of insect specimens: SISBIO authorization Nr. 45742-1, 45742-7 and 45742-10, CNPq process nº 01300.004320/2014-21 and 01300.0013848/2017-33, IBAMA Nr. 14BR016151DF and 20BR035212/DF). This research was supported by funding from the German Science Foundation (DFG) Research Grants FL1051/1-1 and KA2846/6-1.

Publisher Copyright:
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