Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae.

Lance E Keller,Jan-Willem Veening,Jun Kurushima,Anne-Stéphanie Rueff

doi:10.3390/genes10050394

Lance E Keller, Jan-Willem Veening + Show 2 more

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https://doi.org/10.3390/genes10050394

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Journal: Genes	Publication Date: May 22, 2019
Citations: 33	License type: CC BY 4.0

Affiliation: University of Lausanne

Abstract

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

Highlights

Infectious disease is one of the most common causes of death worldwide and is an object of intense scientific study [1]
We have developed and tested three new Pneumococcal Engineering Platform (PEP) vectors, pPEPX, pPEPY, and pPEPZ, for their ability to be transformed into S. pneumoniae, compatibility for co-expression, and frequency of occurrence in pneumococcal isolates
Induction of all strains containing an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter were activated with 1 mM of IPTG for 1 h before visualization by microscopy

Summary

Introduction

Infectious disease is one of the most common causes of death worldwide and is an object of intense scientific study [1]. A powerful tool into the research of human pathogens is the ability to manipulate the pathogen in a desired manner to observe the outcome. These manipulations can take many forms; gene deletions, genetic complementation, gene mutations, or controlled expression. To aid in alteration of a target genome, numerous methods have been developed over the years with varying success depending on the targeted pathogen [2]. Tool development in model organisms tend to be over represented compared to other species, with bacterial human pathogens having fewer options. While the study of model organisms has led to great advances in understanding the nature of diverse bacterial functions, they are not useful for pathogen specific processes.

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