Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study.
Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth.
Putignani
Lorenza
L
Unità di Microbiologia Molecolare, Istituto Nazionale per le Malattie Infettive I.R.C.C.S. Lazzaro Spallanzani, Via Portuense 292, 00149 Roma, Italy.
Ambrosi
Cecilia
C
Ascenzi
Paolo
P
Visca
Paolo
P
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting / Q000379:methods; D007074:Immunoglobulin G / Q000276:immunology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides; D019151:Peptide Library; D010860:Pigments, Biological / Q000096:biosynthesis / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D005290:Ferric Compounds; D007074:Immunoglobulin G; D009842:Oligopeptides; D019151:Peptide Library; D010860:Pigments, Biological; D011994:Recombinant Proteins; C042453:pyoverdin; D006899:Mixed Function Oxygenases; C059419:ornithine N5-oxygenase