Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

Abstract

bLeptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n ‫؍‬ 11), transport (n ‫؍‬ 6), membrane structure (n ‫؍‬ 5), stress response (n ‫؍‬ 2), DNA damage repair (n ‫؍‬ 1), and other processes (n ‫؍‬ 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

“…These data led us to suggest the essential role of DnaK protein in L. biflexa . Accordingly, random transposon libraries in Leptospira failed to recover DnaK-deficient strains 9,10 , strengthening the necessity of this gene. Furthermore, in several other bacteria, DnaK is essential for cell growth not only at elevated temperatures but also under optimal conditions, although the reasons for its crucial role are still unclear 75 .…”

“…have expanded in the past years, but are still limited. Random transposon insertion libraries have been performed in both pathogenic 9 and saprophyte strains 10 . Although these studies have shed light on various gene functions and their role in pathogenesis and basic biology 52–58 , screening libraries of mutants is laborious, and in most cases, there is no evidence of the expected phenotype, since the products of several leptospiral genes are assigned as hypothetical proteins 59,60 .…”

Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

“…The TALE ␤gal gene was digested with NdeI and XbaI, purified, and inserted between the NdeI and SpeI restriction sites of pCRPromFlgB (13) to generate a transcriptional fusion between the TALE gene and the Borrelia burgdorferi flgB promoter. The DNA fragment containing the transcriptional fusions was released by PvuII digestion and cloned into the SmaI site of the E. coli-L. biflexa shuttle vector pMAT (14) to generate plasmid ptale ␤gal , which replicates in L. biflexa. Another TALE construct, named the TALE lig gene, was designed to anneal to the sequence 5=-TCCAATAAATCTTAAGAGA-3=, which is located in homologous promoter regions of ligA and ligB in L. interrogans 170 bp upstream of position ϩ1 (15).…”

Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C

“…Despite the recent advances for genetic manipulation of many microbial species, targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. Gene disruption in pathogenic leptospires relies on random transposon insertion or targeted genetic knockout by infrequent homologous recombination events, coupled with weeks waiting for the growth and selection of colonies on agar media [7][8][9] .To overcome the double recombination requirement for allelic exchange, episomal targeted gene silencing techniques are emerging as an alternative and efficient approach for assessing gene function; the prokaryote immunity type II CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR associated)…”