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Relaxed Specificity of the R1162 Nickase: a Model for Evolution of a System for Conjugative Mobilization of Plasmids. Eric C. Becker, 2003.The primary DNA processing protein for conjugative mobilization of the plasmid R1162 is the transesterase MobA, which acts at a unique site on the plasmid, the origin of transfer (oriT) . Both MobA and oriT are members of a large family of related elements that are widely distributed among bacteria . Each oriT consists of a highly conserved core and an adjacent region that is required for binding by its cognate MobA . The sequence of the adjacent region is important in determining the specificity of the interaction between the Mob protein and the oriT DNA . However, the R1162 MobA is active on the oriT of pSC101, another naturally occurring plasmid . We show here that MobA can recognize oriTs having different sequences in the adjacent region and, with varying frequencies, can cleave these oriTs at the correct position within the core . Along with the structure of the oriTs themselves, these characteristics suggest a model for the evolution of this group of transfer systems . Impact of Specific pbp5 Mutations on Expression of ß-Lactam Resistance in Enterococcus faecium. Louis B. Rice, 2004.We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E . faecium D344SRF . Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations . The levels of resistance were amplified when some mutations were present in combination . In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all ß-lactams . Affinity for penicillin generally correlated with ß-lactam MICs for the mutants, but these associations were not strictly proportional . Presence of a Characteristic D-D-E Motif in IS1 Transposase. Shinya Ohta, 2002.Transposases encoded by various transposable DNA elements and retroviral integrases belong to a family of proteins with three conserved acidic amino acids, D, D, and E, constituting the D-D-E motif that represents the active center of the proteins . IS1, one of the smallest transposable elements in bacteria, encodes a transposase which has been thought not to belong to the family of proteins with the D-D-E motif . In this study, we found several IS1 family elements that were widely distributed not only in eubacteria but also in archaebacteria . The alignment of the transposase amino acid sequences from these IS1 family elements showed that out of 14 acidic amino acids present in IS1 transposase, three (D, D, and E) were conserved in corresponding positions in the transposases encoded by all the elements . Comparison of the IS1 transposase with other proteins with the D-D-E motif revealed that the polypeptide segments surrounding each of the three acidic amino acids were similar . Furthermore, the deduced secondary structures of the transposases encoded by IS1 family elements were similar to one another and to those of proteins with the D-D-E motif . These results strongly suggest that IS1 transposase has the D-D-E motif and thus belongs to the family of proteins with the D-D-E motif . In fact, mutant IS1 transposases with an amino acid substitution for each of the three acidic amino acids possibly constituting the D-D-E motif were not able to promote transposition of IS1, supporting this hypothesis . The D-D-E motif identified in IS1 transposase differs from those in the other proteins in that the polypeptide segment between the second D and third E in IS1 transposase is the shortest, 24 amino acids in length . Because of this difference, the presence of the D-D-E motif in IS1 transposase has not been discovered for some time .
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