A characteristic feature of all the HmuY homologues identified in

A characteristic feature of all the HmuY homologues identified in this study is biofilm

formation. However, although we found several putative HmuY homologues in a broad range of bacteria, the similarity of the amino-acid sequences of HmuY from Porphyromonas and other species was low (5-47%) (see Additional file 1). Only between HmuY proteins encoded within Porphyromonas species was the similarity higher (24-100%) (see Additional file 1). In addition, only P. gingivalis strains possess both histidines engaged in heme coordination selleck kinase inhibitor [20, 21]. Here we also demonstrated that antibodies against purified HmuY raised in rabbits were highly specific and recognized only this antigen in P. gingivalis A7436 and W83 whole-cell lysates compared with a P. gingivalis hmuY deletion mutant strain (TO4) (figure 1), E. coli, or Bacteroides fragilis whole-cell lysates (data not shown). Figure 1 Analysis of HmuY protein in P. gingivalis cell. Detection of HmuY protein in whole-cell lysates of the wild-type W83 and A7436 strains and the hmuY deletion learn more mutant (TO4) strain was performed by SDS-PAGE and Coomassie Brilliant Blue G-250 staining (A) or Western blotting using rabbit anti-HmuY antibodies

and chemiluminescence staining (B). Hm, bacteria grown in basal medium supplemented with hemin; DIP, bacteria grown in basal medium supplemented with dipyridyl for the 1st, 2nd, and 3rd passages. HmuY is exposed on the surface of P. gingivalis cells The N terminus of HmuY shares characteristic features of classical lipoproteins, possessing a signal peptide sequence cleaved off by the signal peptidase II [19, 32]. After removal of the signal peptide, the α-amino group of the N-terminal cysteine is acylated, yielding

a mature lipoprotein. Although HmuY association with the outer membrane of the P. gingivalis cell was previously demonstrated [17, 19, 33], the orientation of the protein in the outer membrane was not examined. Bacterial lipoproteins may be located at the cell surface or directed into the periplasmic space. We hypothesized previously that HmuY functions as an external protein 4��8C [21]. To determine whether HmuY is surface exposed, the proteinase K accessibility assay was employed using the P. gingivalis A7436 and W83 wild-type strains. Upon incubation with proteinase K of intact cells grown under low-iron/heme conditions, most of the HmuY was not degraded (figure 2A). A similar effect was observed when P. gingivalis cells grown under high-iron/heme conditions and E. coli cells over-expressing membrane-associated HmuY were examined (data not shown). It is likely that HmuY may be partially protected by the cell wall, similar to other lipoproteins [34], or resistant to proteinase K digestion. The latter is highly possible since we previously demonstrated that HmuY is resistant to the proteolytic action of trypsin and gingipains [21].

Comments are closed.