PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus

Abstract

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al., 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In Streptococcus uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus, and Lactococcus) of medical, veterinary, and industrial significance.

An update on environmental mastitis: Challenging perceptions

“…Strain‐specific virulence has also been documented for E. faecium (Petersson‐Wolfe et al., ). Potential virulence genes underpinning such differences have not been studied in enterococci, but the genomic tools that have been developed to study virulence factors of S. uberis mastitis could provide insight into the functional genomics of other GPCN species (Blanchard et al., ). As for coliforms, host characteristics affect the outcome of GPCN infections: cows in early lactation responded differently to E. faecium challenge than those in late lactation, and a S. uberis strain that largely failed to cause infection in mid‐lactation animals had been isolated from CM at parturition (Petersson‐Wolfe et al., ; Tassi et al., ).…”

Defining the ABC of gene essentiality in streptococci

“…The precise details of these methods: TraDIS, Tn-seq, HITS, INSeq and PIMMS vary from one another [10–14], yet all produce similar end-point data [15]. Each technique employs a transposon delivery vector to produce a library of random transposition mutants within the bacterial genome.…”

“…The increased accessibility of next-generation sequencing (NGS) technologies has facilitated the development of a variety of transposon-genome junction sequencing techniques, which combine dense mutant libraries and sequencing to identify essential bacterial genomes and assign gene function. The precise details of these methods: TraDIS, Tn-seq, HITS, INSeq and PIMMS vary from one another [ 10 – 14 ], yet all produce similar end-point data [ 15 ]. Each technique employs a transposon delivery vector to produce a library of random transposition mutants within the bacterial genome.…”

“…Exposure of mutant libraries to varying experimental conditions, however, enables the relative fitness and conditional essentiality of each gene to be determined. In recent years, a range of essential bacterial genomes have been published using transposon directed sequencing methods [ 10 , 11 , 14 , 16 – 21 ]. Interrogating genomes in this way provides an unprecedented insight into genome-wide fitness, especially when libraries are subjected to disease relevant conditions either in vitro [ 10 , 16 , 22 ] or in vivo [ 23 – 27 ].…”

“…Here, we present a transposon directed insertion-site sequencing (TraDIS) system which is conducted using standard Illumina sequencer protocols. Dense mutant libraries utilising the plasmid pGh9 carrying the insertion element IS S1 (pGh9:IS S1 ) [ 28 ], have previously been utilised with success in Streptococcus uberis ( S. uberis ) [ 14 ]. We modified pGh9:IS S1 within the 5’ terminal of IS S1 to create six barcoded transposons.…”