High-molecular-weight protein 2 of Yersinia enterocolitica is homologous to AngR of Vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis

Abstract

The iron-regulated irp2 gene is specific for the highly pathogenic Yersinia species and encodes high-molecular-weight protein 2 (HMWP2). Despite the established correlation between the presence of HMWP2 and virulence, the role of this protein is still unknown. To gain insight into the function of HMWP2, the entire coding sequence and the promoter of irp2 were sequenced. Two putative -35 and -10 promoter sequences were identified upstream of a large open reading frame, and two potential Fur-binding sites were found overlapping the second -35 box. The large open reading frame is composed of 6,126 nucleotides and may encode a protein of 2,035 amino acids (ca. 228 kDa) with a pI of 5.81. A signal sequence was not present at the N terminus of the protein. Despite the existence of 30 cysteine residues, carboxymethylation prevented the formation of most if not all disulfide bonds that otherwise occurred when the cells were sonicated. The protein was composed of three main domains: a central region of ca. 850 residues, bordered on each side by a repeat of 550 residues. A high degree of identity (44.5%) was found between HMWP2 and the protein AngR of Vibrio anguillarum. The central part of HMWP2 (after removal of a loop of 337 residues) also displayed significant homology with proteins belonging to the superfamily of adenylate-forming enzymes and, like them, possessed a putative ATP-binding motif that is also present in AngR. In addition, HMWP2 shared with the group of antibiotic and enterochelin synthetases a potential amino acid-binding site. Six consensus sequences defining the superfamily and four defining the family of synthetases were derived from the multiple alignment of the 30 sequences of proteins or repeated domains. A phylogenetic tree that was constructed showed that HMWP2 and AngR are in a family composed of Lys2, EntF, and the tyrocidine, gramicidin, and Beta-lactam synthetases. This finding suggests that HMWP2 may participate in the nonribosomal synthesis of small biologically active peptides.

Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality

“…The osmotic shock procedure was that of LaVallie et af. (1993). The plasmolysing solution (SET) consisted of 20% (w/v) sucrose, 25 mM EDTA.Na, and 20 mM Tris/HCl, pH 8-0.…”

Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF, are induced by extracellular pyochelin in Pseudornonas aeruginosa

“…The greatest similarity was found to the High-Molecular-Weight Protein 2 (HMWP2), the irp2 gene product of Yersinia enterocolitica (37 and 31 '/o identical amino acids in the full length PchE and PchF, respectively) and to the AngR protein of Vibrio anguillarurn (31 and 27 % identical amino acids in PchE and PchF, respectively). HMWP2 and AngR are involved in non-ribosomal biosynthesis of the thiazoline-containing siderophores yersiniabactin and anguibactin, respectively (Guilvout et al, 1993;Tomalsky et al, 1993;Bearden et al, 1997;Pelludat et al, 1998).…”

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

“…Several consensus motifs associated with these activities are present in different peptide synthetases (19), and all of these are present in PvdD (Fig. 3).…”