Construction of Fluorescent Pneumococci for In Vivo Imaging and Labeling of the Chromosome

Advances in fluorescence imaging techniques and development and optimization of fluorescent proteins recent years have made major impacts on different fields of pneumococcal research. This chapter provides methodology for construction of fluorescent pneumococcal strains using fusions to DNA-binding proteins. By expressing fluorescent proteins fused to HlpA, a pneumococcal nucleoid binding protein, brightly fluorescent pneumococci are generated. HlpA fusions may be used both for in vivo imaging of pneumococci as well as for marking the nucleoid in cell biology studies. Furthermore, it also explains how to construct strains for imaging of specific chromosomal loci in pneumococci, using a heterologous ParBS system.