Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

Abstract

cWhole-genome fitness analysis in microbes that uses saturating transposon mutagenesis combined with massively parallel sequencing (Tn-seq) is providing a measure of the contribution of each gene to a given growth condition. With this technique, gene fitness profiles and essential genes are discovered by simultaneous analyses of whether the absence of each gene product alters the growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure to simplify and shorten the process by including delivery of the transposon through conjugation and liquid culture enrichment of the mutant pool, creating transposon liquid enrichment sequencing (TnLE-seq). To illustrate the success of these modifications and the robustness of the procedure, analyses of gene fitness of two cultures of the strictly anaerobic bacterium Desulfovibrio vulgaris Hildenborough were performed, with growth on lactate as the electron donor and sulfate as the electron acceptor. These data demonstrate reproducibility and provide a base condition for analysis of fitness changes in deletion mutants and in various growth conditions. The procedural modifications will facilitate the application of this powerful genetic analysis to microbes lacking a facile genetic system. Pilot studies produced 2.5 ؋ 10 5 and 3.4 ؋ 10 5 unique insertion mutants in the anaerobe Desulfovibrio vulgaris Hildenborough grown under typical laboratory conditions in rich medium. These analyses provided two similar high-resolution maps of gene fitness across the genome, and the method was also applied to growth in minimal medium. These results were also compared to the coverage obtained with a ca. 13,000-member cataloged transposon library constructed by sequencing transposon insertion sites in individual mutants.

A Post-Genomic View of the Ecophysiology, Catabolism and Biotechnological Relevance of Sulphate-Reducing Prokaryotes

“…Several modifications to the Tn-seq procedures have been developed that have been shown to be advantageous for application of gene fitness studies to D. vulgaris Hildenborough and merited the coining of the modified name, TnLE-seq (Fels, Zane, Blake, & Wall, 2013). First rather than transforming the SRP with the vector encoding the transposon, conjugation was used to facilitate the generation of the mutant pool.…”

“…17) by conjugation from the Dap À E. coli BW29427 (Doughty et al, 2011) was shown to produce 2.5 À 10 5 unique insertion mutants overnight. Because the Petri plates needed to obtain this number of mutants would be a significant challenge to handle in an anaerobic chamber, the transposon mutants were not identified as colonies but were enriched in liquid cultures with overnight growth (Fels et al, 2013). Because the growth medium for the mutant population lacked (E) Recovered mutants in experimental condition.…”

“…Replicate data, gathered after a 12-month interval, supported the reproducibility of gene fitness determinations of D. vulgaris genome in lactate/sulphate grown cells (Fels et al, 2013). In one of the first applications, gene fitness profiles were obtained for cultures challenged with inhibitory concentrations of NaNO 3 (Korte et al, 2014).…”

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium

“…A description of the library is in preparation. The library has been used in other studies (39)(40)(41).…”

Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri

“…The constructed mutagenic plasmid was then isolated and transformed into an Escherichia coli strain (WM3064) expressing the transfer function of RP4 (34), containing a dapA deletion, and capable of conjugation with RCH2. Following conjugation (35), RCH2 exconjugants were selected with 50 g kanamycin/ml. Putative exconjugants were initially screened to check their sensitivity to spectinomycin (100 g/ml) so that no kanamycin-resistant isolates containing the targeted gene (resulting from a single recombination event and therefore containing the spectinomycin resistance gene) were carried forward.…”