In the present work I have developed and characterized an in vivo selection system to investigate molecular evolution. The essential gene that codes for adenylate kinase (AK; EC 2.4.7.3) in the gram-positive moderate thermophile Geobacillus stearothermophilus was replaced through homologous recombination with its counterpart from the mesophilic Bacillus subtilis. PCR, DNA sequencing and Southern analysis confirmed proper gene replacement and preservation of neighboring genes in the recombinant strain. Recombinant cells (NUB3621-R:ThEV) displayed a temperature sensitive phenotype, with a highest growing temperature almost 20°C lower than that of wild-type cells (56°C vs 75°C). The temperature sensitive phenotype in recombinant cells was linked to a disruption of adenylate homeostasis at high temperatures, secondary to B. subtilis AK heat inactivation, as shown by enzyme activity assays, temperature denaturation profiles and adenylate level measurements. Evolution of a single gene, B. subtilis adk, was investigated by steady-state growth of the recombinant strain from 55 to 70°C in a turbidostat. The temporal characteristics of B. subtilis adk evolution were probed by DNA sequence analysis at various temperatures. The appearance of more fit strains from pre-existing genetic variation, rapid extinctions generated by clonal interference and selection coupled to a strong dependence on historical context and chance events resulted in a punctuated pattern of evolution. The organism fitness at different temperatures could be linked to its expressed AK variant and allowed the investigation of the biochemical and structural basis of adaptation. Heat denaturation and enzyme activity studies showed that all isolated AK mutants were more stable than the wild-type protein and were responsible for the punctuated pattern of evolution detected during the population analyses. The atomic structure of one of the isolated AK variants (Q199R) revealed that its increased thermostability is due to unique electrostatic interactions absent in the wild-type structure. Taken together our population, biochemical and structural analysis suggests that natural selection observed at the molecular level is guided by the same principles that act at the organismal level.