Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus suis

Abstract

HPr is a low-molecular-mass phosphocarrier protein of the bacterial phosphoenolpyruvate (PEP) :sugar phosphotransferase system (PTS) found in the cytoplasm or associated with the inner surface of the cytoplasmic membrane. Treatment of Streptococcus suis cells with a SorvaII Omnimixer, a technique used to extract cell surface components, resulted in the extraction of a major protein with a molecular mass of 9 kDa. Several lines of evidence suggested that this protein was HPr: (i) the 5. suis protein showed homology over the first 35 N-terminal amino acid residues with the HPrs of Streptococcus salivarius and Streptococcus mutans, including the signature sequence for the site of PEP-dependent phosphorylation; (ii) it cross-reacted with the 5. salivarius anti-HPr antibody preparation; (iii) it could be phosphorylated by enzyme I at the expense of PEP, and by a membrane-associated kinase a t the expense of ATP; and (iv) it possessed phosphocarrier activity when used as a source of HPr in an in vitro PTS assay. The data suggested that a portion of the cellular HPr is associated with the external cell surface in S. suis, a result that was confirmed by immunogold electron microscopy. The cellular HPr of 5. suis consisted of two forms that could be distinguished by the presence or the absence of the N-terminal methionine. Amino acid sequence analysis indicated that the cell-surface-associated HPr of S. suis lacked the N-terminal methionine residue.

Identification and Immunogenicity of Group AStreptococcusCulture Supernatant Proteins

“…More than one-half of the culture supernatant proteins that we identified do not have apparent secretion signal sequences and are traditionally considered to be cytosolic proteins involved in intracellular processes. However, several of these proteins have been previously reported to be located extracellularly in streptococci, including histone-like protein (44), glyceraldehyde-3-phosphate dehydrogenase (37,38,51,52), ␣-enolase (39), arginine deiminase (antitumor glycoprotein) (9,23), and phosphocarrier protein (11). Glycolytic enzymes also have been detected in association with the cell surfaces of several other microbial pathogens (13,32).…”

Catabolite Control Protein A (CcpA) Contributes to Virulence and Regulation of Sugar Metabolism in Streptococcus pneumoniae

“…HPr, which is one of the chief players in PTS-mediated sugar uptake, is normally a cytoplasmic protein. It has been shown to also localize to the surface in Streptococcus suis cells (18). Cell surface-associated HPr, although retaining function, was shown to be devoid of the N-terminal methionine residue, a modification that may lead to its altered location.…”

The EIIABManPhosphotransferase System Permease Regulates Carbohydrate Catabolite Repression inStreptococcus gordonii

“…All samples were treated at 100°C for 5 min to eliminate His15-phosphorylated HPr prior to resolving the proteins on a 12.5% nondenaturing polyacrylamide gel. The doublets of both HPr (top) and HPr-Ser-P (bottom) represent the full-length protein and HPr without the N-terminal methionine (16,48). The antisera, kindly provided by Christian Vadeboncouer, was raised against purified HPr from Streptococcus salivarius and used as previously described (55).…”

“…Therefore, an anti-HPr antiserum was used in Western blots to detect the levels of seryl-phosphorylated and unphosphorylated HPr in the wild-type and various mutant backgrounds. Lysates were prepared from cells growing exponentially in BHI and separated by nondenaturing polyacrylamide gel after treatment at 100°C for 5 min to remove any phosphate from His15, as described elsewhere (16,48,55). Significantly higher levels of HPr-Ser-P were produced in the strain lacking CcpA (Fig.…”