A stretch of hydrophobic amino acids between residues 10 and 32 could serve to anchor the XpsN protein in the cytoplasmic membrane. tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis exposed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)6-tagged XpsD protein from the metallic affinity chromatography column. All observations suggested the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with numerous truncated XpsD proteins exposed the PTZ-343 C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved Rabbit Polyclonal to ATG16L2 in complex formation between the two. The type II secretion pathway is definitely common among gram-negative bacteria (48, 49). It is also referred to as the main terminal branch of the general secretory pathway (38). In all cases, the secreted proteins possess a typical N-terminal transmission sequence, which is definitely processed while becoming exported across the inner membrane. Between 11 and 14 genes are required for the second step of the secretion pathway. Each encodes a protein that has amino acid sequence homology to different degrees with other proteins of the same family. An outer membrane protein belonging to the GspD protein family has been demonstrated to form multimeric complex (2, 7, 25, 31) and suggested to serve as a gated secretion channel (33). A second outer membrane-associated protein, GspS (PulS in the case of by Filloux et al. (15). The two gene clusters of and share strong sequence homology and gene corporation. However, the gene is definitely absent in the former. Neither is the gene encoding the GspN homologue present in the gene cluster, while it is definitely encoded by most other type II secretion gene clusters (19, 40, 41, 51). Pairwise amino acid sequence alignments among the GspN proteins revealed the highest sequence identities of 34.7% between the PulN protein of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P15753″,”term_id”:”131602″,”term_text”:”P15753″P15753) and the OutN protein of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P31710″,”term_id”:”399823″,”term_text”:”P31710″P31710) and 39% between the ExeN protein of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P41852″,”term_id”:”1170072″,”term_text”:”P41852″P41852) and the EpsN protein of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P45784″,”term_id”:”12644271″,”term_text”:”P45784″P45784). In contrast, with the same assessment guidelines (blosum 62; space creation of 8 and space extension of 2 [18]), the sequence identity between PulD of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P15644″,”term_id”:”131592″,”term_text”:”P15644″P15644) and OutD of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P31701″,”term_id”:”2506491″,”term_text”:”P31701″P31701) is definitely 72.5% and that between ExeD of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P31780″,”term_id”:”1170050″,”term_text”:”P31780″P31780) and EpsD of (accession no. “type”:”entrez-protein”,”attrs”:”text”:”P45779″,”term_id”:”11182423″,”term_text”:”P45779″P45779) is definitely 55.8%. These analyses suggest that the amino acid sequence of the GspN protein is probably not as well conserved as that of the GspD protein. Upstream of the gene in pv. campestris, we have previously recognized two open reading frames (ORFs) (ORF1 and ORF2 [20]). ORF2 could encode a protein of 261 or 257 amino acid PTZ-343 residues. We tentatively assigned the 1st in-frame ATG as the initiation codon (Fig. ?(Fig.1),1), because we located a putative Shine-Dalgarno sequenceGGAGG6 nucleotides upstream of it. Searching in the GenBank database with filters to exclude input sequences with low difficulty or repeat areas (1) retrieved no additional protein. Its N-terminal amino acid sequence resembles a likely membrane-anchoring sequence (Fig. ?(Fig.1).1). Such analysis raised our PTZ-343 interest to determine the significance of ORF2 in extracellular protein secretion in pv. campestris. We designate it the gene for the following reasons. Its location in the gene cluster and the expected molecular excess weight of its putative protein product are similar to those of all genes of the type II protein secretion pathway. Upstream of ORF2, we have recognized nine ORFs (unpublished results; accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”L02630″,”term_id”:”73916934″,”term_text”:”L02630″L02630 and “type”:”entrez-nucleotide”,”attrs”:”text”:”M81648″,”term_id”:”155388″,”term_text”:”M81648″M81648), the deduced amino acid sequences of which have sequence homologies to numerous extents, as well as the same gene order, with the genes of genes by Dums et al. (12). Furthermore, as discussed previously, the amino acid sequence homology among all known GspN proteins is not.