Deletion and Substitution Analysis of the Escherichia coli TonB Q160 Region

ABSTRACT The active transport of iron siderophores and vitamin B12 across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBΔ7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBΔ7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.

Microglial expression of peptidylarginine deiminase 2 in the prenatal rat brain

Peptidylarginine deiminases (PADs) are Ca2t+-dependant post-translational modification enzymes that catalyze the citrullination of protein arginyl residues. PAD type 2 (PAD2) is thought to be involved in some processes of neurodegeneration and myelination in the central nervous system. In this study, we found PAD2-positive cells in rat cerebra in 19-to 21-day old embryos, i.e. at a developmental stage well before myelination begins. Most of the cells were microglial marker-positive cells found mainly in the prospective medulla, and others were microglial marker-negative cells found mainly in the prospective dentate gyrus of the hippocampus. The former seemed to be in an activated state as judged by morphological criteria. The specificity of the enzyme activity, immunoblotting and reverse transcriptase-polymerase chain reaction analyses revealed that these cells expressed PAD2 and not PAD1, PAD3 or PAD4. Our data is indicative of microglial expression of PAD2 in the prenatal developing cerebrum.