Fig 4.

Pfo expression in Streptococcus pneumoniae. (A) Amino acid alignment of Ply (top) and Pfo (bottom). Sequence homology does not begin until the third amino acid of Ply; therefore, fusions were made at this amino acid. The arrowhead indicates the site of signal peptidase cleavage in Pfo. The Pfo sequence not in bold was excluded in the Pfo signal sequence-less construct, thus deleting the Pfo signal peptide and six amino acids after the signal peptidase cleavage that share no homology to Ply. (B) Expression of signal sequence-less Pfo in S. pneumoniae. Signal sequence-less Pfo is expressed in S. pneumoniae, and a small amount localizes to the cell wall. The Pfo signal sequence-less allele (pfoNoSS) was used to replace ply at the native locus (Δply::pfoNoSS). This strain and a negative-control strain in which the ply deletion was replaced with a spectinomycin resistance gene (Δply::spc) were grown to mid-exponential phase, fractionated into supernatant, cell wall, and protoplast compartments, and assayed for the presence of Ply and CodY by Western blotting with anti-Pfo and anti-CodY antibodies. Equal cell equivalents were loaded on the gel.