Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF

Abstract

The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.

Parallel genomics uncover novel enterococcal-bacteriophage interactions

“…To identify genetic determinants that confer phage resistance in E. faecalis , an E. faecalis OG1RF transposon library consisting of 6,829 unique mutants was screened by s equence-defined mar iner technology tr ansposon seq uencing (SMarT TnSeq) (33). 10 7 CFU of logarithmically growing E. faecalis TnSeq library pool was plated on solid media in the absence and presence of phage VPE25 at a multiplicity of infection (MOI) of 0.1.…”

“…Cells from the input library prior to plating and cells plated on plates containing no phage were used as controls. Tn insertions in E. faecalis genomic DNA were sequenced as described by Dale et al (33). Sequencing reads were mapped to the E. faecalis OG1RF genome to identify bacterial mutants with altered phage sensitivity.…”

“…Therefore, to fill this gap and further define the molecular underpinnings of enterococcal-phage interactions, we have taken a global genomics approach to identify enterococcal factors critical for productive infection by the lytic phage VPE25 (32). To identify bacterial genes essential for VPE25 infection, we screened a low-complexity transposon (Tn) mutant library of E. faecalis OG1RF for phage resistance (33). In addition to the known VPE25 receptor (32), transposon sequencing revealed novel E. faecalis genes necessary for phage adsorption and optimum intracellular phage DNA replication and transcription.…”

Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

“…Recent technological advances have vastly lowered the cost and effort required to order random transposon insertion libraries 15,16 . Pooling strategies that combine cultures prior to sequencing and then use computational methods to identify the positions of individual strains can reduce the number of samples that need to be processed by two orders of magnitude 5,7,10,16,17 . The strategy behind various pooling protocols can be separated into two parts.…”

“…Current sequencing approaches for transposon insertion libraries use a flavor of transposon insertion sequencing (Tn-seq/INSeq/ST-PCR). Because these sequencing approaches map transposon insertions for every individual pool, they remain laborious, often requiring multiple processing steps for each individual pool 7,10,15,16 .…”