Juan, P.-A. | Search bioRxiv

bioRxiv2020

Transposon-insertion sequencing in a clinical isolate of Legionella pneumophila identifies essential genes and determinants of natural transformation

Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdown horizontal gene transfer events. Transposon-insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of difficulty to obtain a high-saturation transposon insertion library. Indeed, we found that ST1 isolates, to which belong the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we therefore used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor Type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. ImportanceLegionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. L. pneumophila is found in man-made and freshwater environments which are the causing source of the infection. The environmental life traits of L. pneumophila, such as its abilities to form biofilms, resist biocides and unicellular predators, are essential to its ability to accidentally infect humans. A comprehensive identification of the genetic basis of these life traits could be obtained through the use of transposon-insertion sequencing. Yet, this powerful approach, had not been fully implemented in L. pneumophila. Here we described the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step towards identifying the genetic basis of the many life traits of this environmental and pathogenic species.

microbiology↗

bioRxiv2019

Diverse conjugative elements silence natural transformation in Legionella species

Natural transformation, i.e. the uptake of DNA and its stable integration in the chromosome, is a major mechanism of horizontal gene transfer and is common in bacteria. The vast majority of bacterial genomes carry the specific genes involved in natural transformation, yet only a fraction of species are deemed naturally transformable. This is typically explained by the inability of standard laboratory conditions to induce this phenotypic trait. However, even when the inducing conditions are known, large intraspecific variations have been reported. In this study, we investigated the conservation and distribution of natural transformability in the human pathogen Legionella pneumophila. Using a panel of 113 clinical isolates, we found that natural transformability is relatively conserved but shows large variations inconsistent with the phylogeny. By conducting a genome-wide association study (GWAS) we identified the conjugative plasmid pLPL as a source of these intraspecific variations. We further show that the plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination, comEC, comEA, comF and comM. We identified a plasmid-encoded small RNA (sRNA), RocRp, as solely responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded RocR which controls the transient expression of the DNA uptake system. We show that RocRp can take over the function of RocR, by acting as a substitute, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species, suggesting that silencing natural transformation is beneficial to these genetic elements. We propose that transformation-interfering factors are frequent genetic cargo of mobile genetic elements, accounting for intraspecific variations in natural transformation but also responsible for the apparent non-transformability of some species.

microbiology↗