Repeat numbers at nine variable-number tandem-repeat (VNTR) loci were determined for 177 isolates of Pseudomonas aeruginosa representing 77 strains distinguished by pulsed-field gel electrophoresis (PFGE). Eight loci provided for discrimination similar to that provided by PFGE, with variation at the ninth locus (ms61) sometimes allowing discrimination within a PFGE-defined type. The Liverpool and Midlands 1 strains, which are common among patients with cystic fibrosis in the UK, could be unambiguously identified by their characteristic VNTR profiles. In rare cases, the repeat number at the ninth locus alone provided discrimination among isolates that were distinct according to PFGE. In each case, the two isolates shared the same bla(OXA-50-like) allele and belonged to the same oprD sequence type group, supporting the VNTR results in suggesting that they are similar.

The engineered three-helix bundle, UVF, is thermostabilized entropically due to heightened, native-state dynamics. However, it is unclear whether this thermostabilization strategy is observed in natural proteins from thermophiles. We performed all-atom, explicit solvent molecular dynamics simulations of two three-helix bundles from thermophilic H. butylicus (2lvsN and 2lvsC) and compared their dynamics to a mesophilic three-helix bundle, the Engrailed homeodomain (EnHD). Like UVF, 2lvsC had heightened native dynamics, which it maintained without unfolding at 100 C. Shortening and rigidification of loops in 2lvsN and 2lvsC and increased surface hydrogen bonds in 2lvsN were observed, as is common in thermophilic proteins. A buried disulfide and salt bridge in 2lvsN and 2lvsC, respectively, provided some stabilization, and addition of a homologous disulfide bond in EnHD slowed unfolding. The transferability and commonality of stabilization strategies among members of the three-helix bundle fold suggest that these strategies may be general and deployable in designing thermostable proteins.

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