title: Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Mycoplasma gallisepticum.
abstract: The significance of the amino acid adjacent to the amino terminal cysteine of lipoproteins, the +2 amino acid, has been well documented in E. coli and there have also been limited studies on Gram-positive bacteria. In this study we investigated whether there was any preference for specific residues and any targeting role attributable to different residues following the cysteine at the amino terminus in lipoproteins of Mycoplasma gallisepticum. There were found to be distinct preferences in this position that vary considerably from the preferences seen in Gram-positive and Gram-negative bacteria. The effect of different amino acids at the +2 position was studied using the pTAP vector, which has been shown to express PhoA as a lipoprotein. Replacement of the threonine at the +2 position in the PhoA lipoprotein with hydrophobic amino acids resulted in higher levels of expression of alkaline phosphatase, while replacement with hydrophilic amino acids resulted in lower levels of expression of alkaline phosphatase. Changes in the +2 amino acid did not appear to alter export of the PhoA lipoprotein to the membrane fraction, but a difference was seen in susceptibility to proteolysis in PhoA lipoproteins with differing +2 amino acids. This is the first study to examine the role of the +2 amino acid in mycoplasma lipoproteins and establish a difference between M. gallisepticum and Gram-positive and Gram-negative bacteria and will assist in optimization of the design of recombinant lipoprotein genes in mycoplasmas for maximal levels of expression and stability on the cell surface.
authors.affiliation: Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Indu S
authors.initials: IS
authors.lastname: Panicker
authors.affiliation: Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Kanci
authors.affiliation: Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Philip F
authors.initials: PF
authors.lastname: Markham
authors.affiliation: Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Glenn F
authors.initials: GF
authors.lastname: Browning
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D045686:Mycoplasma gallisepticum / Q000235:genetics*
chemical_list: D008074:Lipoproteins; D008565:Membrane Proteins; D000469:Alkaline Phosphatase