Cellular localization of choline‐utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

Abstract

SummaryThe molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zincinducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

“…With this tool we showed that S. pneumoniae DivIVA localizes to both the cell division sites and the cell poles (35). We now show that StkP also localizes to the midcell and that this localization pattern depends on its extracellular PASTA domains.…”

“…To determine the localization and dynamics of StkP in living cells, we constructed an N-terminal gfpstkP fusion (Fig. 1A) by using vector pJWV25, which integrates by double-crossover in the chromosome at the nonessential bgaA locus and harbors the zinc-inducible P Zn promoter (35). The resulting construct then was introduced into three closely related well-characterized and widely used S. pneumoniae strains: the encapsulated D39 and nonencapsulated R6 and Rx1 genetic backgrounds (36).…”

“…We previously developed a single-cell toolbox for S. pneumoniae that allowed in vivo protein-localization studies in live pneumococcal cells using a fast-folding variant of GFP (35).…”

Peptide-Regulated Gene Depletion System Developed for Use in Streptococcus pneumoniae

“…Since all S. pneumoniae strains tested so far have been unable to grow on fucose as a sole carbon and energy source, the role of this sugar in pneumococcal metabolism remains unclear (3). An alternative method based on the Zn 2ϩ -inducible promoter P czcD was described recently (7,17). To perform a gene depletion experiment with the Zn 2ϩ system, a DNA cassette containing a selectable antibiotic resistance marker and the gene of interest under the control of the P czcD promoter was inserted into the FIG.…”

Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

“…The pJVW25 plasmid was used to express RrgC, Srt-C1, and Srt-C3 in pneumococcal strains (30). Protein expression was under the control of the zinc-inducible PczcD promoter and required addition of 0.15 mM ZnCl 2 in the liquid medium.…”