Advancing Genetic Tools in Streptococcus pneumoniae.

Haley Echlin,Jason W Rosch

doi:10.3390/genes11090965

Abstract

Streptococcus pneumoniae is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are available to manipulate this pathogen. In this study, we attempted to utilize one such tool, the Sweet Janus cassette, to replace the capsule locus with other capsule loci in our strain background and found that the efficiency of allelic replacement was low and the number of revertant false-positive colonies was high. We determined that the capacity to recombine capsule varied by the initial isolated colony, suggesting that frequency of reversion is dependent on the bacterial clone. Alternative selection markers may further expand the application of Sweet Janus. We created novel cassettes that utilized chlorinated phenylalanine as an alternative counter-selection agent in conjunction with the Janus or Sweet Janus cassette, providing a new dual or triple selection marker. Moreover, we created cassettes that do not require engineered resistance in the background strain, including both single and dual selection markers. We were able to utilize all constructs in allelic replacement of the capsule loci. These novel constructs provide a new means for generating gene deletions in S. pneumoniae that expand experimental applications.

Highlights

Streptococcus pneumoniae is a major human pathogen with high rates of mortality and disease, including sinusitis and otitis media, pneumonia, bacteremia, and meningitis [1]
To maintain an isogenic cell line, we utilized this system to generate an unencapsulated form of our TIGR4 through allelic replacement of the capsule locus with Sweet Janus
Since the Sweet Janus cassette relies upon streptomycin resistance in the background strain, we first generated a streptomycin resistant variant of TIGR4

Summary

Introduction

Streptococcus pneumoniae is a major human pathogen with high rates of mortality and disease, including sinusitis and otitis media, pneumonia, bacteremia, and meningitis [1]. While there are several means of creating genetic deletions, most involve introduction of resistant cassettes as a selectable marker to identify recombinants. Imparting resistance can have drawbacks in multiple forms, including inability to assess drug resistance in mutants due to potential cross-resistance, reduced number of potential combination of gene deletions, potential stress caused by production of resistance marker, and compromising experimental results. To overcome many of these shortcomings, a markerless cassette known as Janus was generated [7]. This cassette yielded the opportunity to study many gene functions in previously unavailable situations through allelic replacement or deletion [8,9,10]

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